Integrated monolithic microfabricated electrospray and liquid chromatography system and method

ABSTRACT

An electrospray device, a liquid chromatography device and an electrospray-liquid chromatography system are disclosed. The electrospray device comprises a substrate defining a channel between an entrance orifice on an injection surface and an exit orifice on an ejection surface, a nozzle defined by a portion recessed from the ejection surface surrounding the exit orifice, and an electrode for application of an electric potential to the substrate to optimize and generate an electrospray; and, optionally, additional electrode(s) to further modify the electrospray. The liquid chromatography device comprises a separation substrate defining an introduction channel between an entrance orifice and a reservoir and a separation channel between the reservoir and an exit orifice, the separation channel being populated with separation posts perpendicular to the fluid flow; a cover substrate bonded to the separation substrate to enclose the reservoir and the separation channel adjacent the cover substrate; and, optionally, electrode(s) for application of a electric potential to the fluid. The exit orifice of the liquid chromatography device may be homogeneously interfaced with the entrance orifice of the electrospray device to form an integrated single system. An array of multiple systems may be fabricated in a single monolithic chip for rapid sequential fluid processing and generation of electrospray for subsequent analysis, such as by positioning the exit orifices of the electrospray devices near the sampling orifice of a mass spectrometer.

This application is a divisional of U.S. application Ser. No. 09/156,507filed Sep. 17, 1998.

FIELD OF THE INVENTION

The present invention relates generally to an integrated miniaturizedchemical analysis system fabricated using microelectromechanical systems(MEMS) technology. In particular, the present invention relates to anintegrated monolithic microfabricated electrospray and liquidchromatography device. This achieves a significant advantage in terms ofhigh-throughput analysis by mass spectrometry, as used, for example, indrug discovery, in comparison to a conventional system.

BACKGROUND OF THE INVENTION

New developments in drug discovery and development are creating newdemands on analytical techniques. For example, combinatorial chemistryis often employed to discover new lead compounds or to create variationsof a lead compound. Combinatorial chemistry techniques can generatethousands or millions of compounds (combinatorial libraries) in arelatively short time (on the order of days to weeks). Testing such alarge number of compounds for biological activity in a timely andefficient manner requires high-throughput screening methods which: allowrapid evaluation of the characteristics of each candidate compound.

The compounds in combinatorial libraries are often tested simultaneouslyagainst a molecular target. For example, an enzyme assay employing acolorimetric measurement may be run in a 96-well plate. An aliquot ofenzyme in each well is combined with tens or hundreds of compounds. Aneffective enzyme inhibitor will prevent development of color due to thenormal enzyme reaction, allowing for rapid spectroscopic (or visual)evaluation of assay results. If ten compounds are present in each well,960 compounds can be screened in the entire plate, and one hundredthousand compounds can be screened in 105 plates, allowing for rapid andautomated testing of the compounds.

Often, however, determination of which compounds are present in certainportions of a combinatorial library is difficult; due to the manner ofsynthesis of the library. For example, the “split-and-pool” method ofrandom peptide synthesis in U.S. Pat. No. 5,182,366, describes a way ofcreating a peptide library where each resin bead carries a uniquepeptide sequence. Placing ten beads in each well of a 96-well plate,followed by cleavage of the peptides from the beads and removal of thecleavage solution, would result in ten (or fewer) peptides in each wellof the plate. Enzyme assays could then be carried out in the platewells, allowing 100,000 peptides to be screened in 105 plates. However,the identity of the peptides would not be known, requiring analysis ofthe contents of each well.

The peptides could be analyzed by removing a portion of solution fromeach well and injecting the contents into a separation device such asliquid chromatography or capillary electrophoresis instrument coupled toa mass spectrometer. Assuming that such a method would takeapproximately 5 minutes per analysis, it would require over a month toanalyze the contents of 105 96-well plates, assuming the method wasfully automated and operating 24 hours a day.

This example illustrates the critical need for a method for rapidanalysis of large numbers of compounds or complex mixtures of compounds,particularly in the context of high-throughput screening. Techniques forgenerating large numbers of compounds, for example through combinatorialchemistry, have been established. High-throughput screening methods areunder development for a wide variety of targets, and some types ofscreens, such as the calorimetric enzyme assay described above and ELISA(enzyme linked immunosorbent assay) technology, are well-established. Asindicated in the example above, a bottleneck often occurs at the stagewhere multiple mixtures of compounds, or even multiple individualcompounds, must be characterized.

This need is further underscored when current developments in molecularbiotechnology are considered. Enormous amounts of genetic sequence dataare being generated through new DNA sequencing methods. This wealth ofnew information is generating new insights into the mechanism of diseaseprocesses. In particular, the burgeoning field of genomics has allowedrapid identification of new targets for drug development efforts.Determination of genetic variations between individuals has opened upthe possibility of targeting drugs to individuals based on theindividual's particular genetic profile. Testing for cytotoxicity,specificity, and other pharmaceutical characteristics could be carriedout in high-throughput assays instead of expensive animal testing andclinical trials. Detailed characterization of a potential drug or leadcompound early in the drug development process thus has the potentialfor significant savings both in time and expense.

Development of viable screening methods for these new targets will oftendepend on the availability of rapid separation and analysis techniquesfor analyzing the results of assays. For example, an assay for potentialtoxic metabolites of a candidate drug would need to identify both thecandidate drug and the metabolites of that candidate. An assay forspecificity would need to identify compounds which bind differentiallyto two molecular targets such as a viral protease and a mammalianprotease.

It would therefore be advantageous to provide a method for efficientproteomic screening in order to obtain the pharmacokinetic profile of adrug early in the evaluation process. An understanding of how a newcompound is absorbed in the body and how it is metabolized can enableprediction of the likelihood for an increased therapeutic effect or lackthereof.

Given the enormous number of new compounds that are being generateddaily, an improved system for identifying molecules of potentialtherapeutic value for drug discovery is also critically needed.

It also would be desirable to provide rapid sequential analysis andidentification of compounds which interact with a gene or gene productthat plays a role in a disease of interest. Rapid sequential analysiscan overcome the bottleneck of inefficient and time-consuming serial(one-by-one) analysis of compounds.

Accordingly, there is a critical need for high-throughput screening andidentification of compound-target reactions in order to identifypotential drug candidates.

Microchip-based separation devices have been developed for rapidanalysis of large numbers of samples. Compared to other conventionalseparation devices, these microchip-based separation devices have highersample throughput, reduced sample and reagent consumption and reducedchemical waste. The liquid flow rates for microchip-based separationdevices range from approximately 1-300 nanoliters (nL) per minute formost applications.

Examples of microchip-based separation devices include those forcapillary electrophoresis (CE), capillary electrochromatography (CEC)and high-performance liquid chromatography (HPLC). See Harrison etal,Science 1993, 261, 859-897; Jacobson etal. Anal. Chem. 1994, 66,1114-1118; and Jacobson etal. Anal. Chem. 1994, 66, 2369-2373. Suchseparation devices are capable of fast analyses and provide improvedprecision and reliability compared to other conventional analyticalinstruments.

Liquid chromatography (LC) is a well-established analytical method forseparating components of a fluid for subsequent analysis and/oridentification. Traditionally, liquid chromatography utilizes aseparation column, such as a cylindrical tube, filled with tightlypacked beads, gel or other appropriate particulate material to provide alarge surface area. The large surface area facilitates fluidinteractions with the particulate material, and the tightly packed,random spacing of the particulate material forces the liquid to travelover a much longer effective path than the length of the column. Inparticular, the components of the fluid interact with the stationaryphase (the particles in the liquid chromatography column) as well as themobile phase (the liquid eluent flowing through the liquidchromatography column) based on the partition coefficients for each ofthe components. The partition coefficient is a defined as the ratio ofthe time an analyte spends interacting with the stationary phase to thetime spent interacting with the mobile phase. The longer an analyteinteracts with the stationary phase, the higher the partitioncoefficient and the longer the analyte is retained on the liquidchromatography column. The components may be detected spectroscopicallyafter elution from the liquid chromatography column by coupling the exitof the column to a post-column detector.

Spectroscopic detectors rely on a change in refractive index,ultraviolet and/or visible light absorption, or fluorescence afterexcitation with a suitable wavelength to detect the separatedcomponents. Alternatively, the separated components may be passed fromthe liquid chromatography column into other types of analyticalinstruments for analysis. The analysis outcome depends upon thesequenced arrival of the components separated by the liquidchromatography column and is therefore time-dependent.

The length of liquid transport from the liquid chromatography column tothe analysis instrument such as the detector is preferably minimized inorder to minimize diffusion and thereby maximize the separationefficiency and analysis sensitivity. The transport length is referred toas the dead volume or extra-column volume.

Capillary electrophoresis is a technique that utilizes theelectrophoretic nature of molecules and/or the electroosmotic flow offluids in small capillary tubes to separate components of a fluid.Typically a fused silica capillary of 100 μm inner diameter or less isfilled with a buffer solution containing an electrolyte. Each end of thecapillary is placed in a separate fluidic reservoir containing a bufferelectrolyte.

A potential voltage is placed in one of the buffer reservoirs and asecond potential voltage is placed in the other buffer reservoir.Positively and negatively charged species will migrate in oppositedirections through the capillary under the influence of the electricfield established by the two potential voltages applied to the bufferreservoirs. Electroosmotic flow is defined as the fluid flow along thewalls of a capillary due to the migration of charged species from thebuffer solution. Some molecules exist as charged species when insolution and will migrate through the capillary based on thecharge-to-mass ratio of the molecular species. This migration is definedas electrophoretic mobility. The electroosmotic flow and theelectrophoretic mobility of each component of a fluid determine theoverall migration for each fluidic component. The fluid flow profileresulting from electroosmotic flow is flat due to the reduction infrictional drag along the walls of the separation channel. This resultsin improved separation efficiency over liquid chromatography where theflow profile is parabolic resulting from pressure driven flow.

Capillary electrochromatography is a hybrid technique which utilizes theelectrically driven flow characteristics of electrophoretic separationmethods within capillary columns packed with a solid stationary phasetypical of liquid chromatography. It couples the separation power ofreversed-phase liquid chromatography with the high efficiencies ofcapillary electrophoresis. Higher efficiencies are obtainable forcapillary electro-chromatography separations over liquid chromatographybecause the flow profile resulting from electroosmotic flow is flat dueto the reduction in frictional drag along the walls of the separationchannel when compared to the parabolic flow profile resulting frompressure driven flows. Furthermore, smaller particle sizes can be usedin capillary electrochromatography than in liquid chromatography becauseno back pressure is generated by electroosmotic flow. In contrast toelectrophoresis, capillary electrochromatography is capable ofseparating neutral molecules due to analyte partitioning between thestationary and mobile phases of the column particles using a liquidchromatography separation mechanism.

The separated product of such separation devices may be introduced asthe liquid sample to a device that is used to produce electrosprayionization. The electrospray device may be interfaced to an atmosphericpressure ionization mass spectrometer (API-MS) for analysis of theelectrosprayed fluid.

A schematic of an electrospray system 50 is shown in FIG. 1. Anelectrospray is produced when a sufficient electrical potentialdifference V_(spray) is applied between a conductive or partlyconductive fluid exiting a capillary orifice and an electrode so as togenerate a concentration of electric field lines emanating from the tipor end of a capillary 52 of an electrospray device. When a positivevoltage V_(spray) is applied to the tip of the capillary relative to anextracting electrode 54, such as one provided at the ion-samplingorifice to the mass spectrometer, the electric field causespositively-charged ions in the fluid to migrate to the surface of thefluid at the tip of the capillary. When a negative voltage V_(spray)applied to the tip of the capillary relative to an extracting electrode54, such as one provided at the ion-sampling orifice to the massspectrometer, the electric field causes negatively-charged ions in thefluid to migrate to the surface of the fluid at the tip of thecapillary.

When the repulsion force of the solvated ions exceeds the surfacetension of the fluid sample being electrosprayed, a volume of the fluidsample is pulled into the shape of a cone, known as a Taylor cone 56which extends from the tip of the capillary. Small charged droplets 58are formed from the tip of the Taylor cone 56 and are drawn toward theextracting electrode 54. This phenomenon has been described, forexample, by Dole et al., Chem. Phys. 1968,49,2240 and Yamashita andFenn, J Phys. Chem. 1984, 88, 4451. The potential voltage required toinitiate an electrospray is dependent on the surface tension of thesolution as described by, for example, Smith, IEEE Trans. Ind. App.1986, IA-22, 527-535. Typically, the electric field is on the order ofapproximately 10⁶ V/m. The physical size of the capillary determines thedensity of electric field lines necessary to induce electrospray.

One advantage of electrospray ionization is that the response for ananalyte measured by the mass spectrometer detector is dependent on theconcentration of the analyte in the fluid and independent of the fluidflow rate. The response of an analyte in solution at a givenconcentration would be comparable; using electrospray ionizationcombined with mass spectrometry at a flow rate of 100 μL/min compared toa flow rate of 100 nL/min.

The process of electrospray ionization at flow rates on the order ofnanoliters per minute has been referred to as “nanoelectrospray”.Electrospray into the ion-sampling orifice of an API mass spectrometerproduces a quantitative response from the mass spectrometer detector dueto the analyte molecules present in the liquid flowing from thecapillary. Thus, it is desirable to provide an electrospray ionizationdevice for integration upstream with microchip-based separation devicesand for integration downstream with API-MS instruments.

Attempts have been made to manufacture an electrospray device whichproduces nanoelectrospray. For example, Wilm and Mann, Anal. Chem. 1996,68, 1-8 describes the process of electrospray from fused silicacapillaries drawn to an inner diameter of 2-4 μm at flow rates of 20nL/min. Specifically, a nanoelectrospray at 20 nL/min was achieved froma 2 μm inner diameter and 5 μm outer diameter pulled fused-silicacapillary with 600-700 V at a distance of 1-2 mm from the ion-samplingorifice of an API mass spectrometer.

Ramsey et al., Anal. Chem. 1997, 69, 1174-1178 describesnanoelectrospray at 90 nL/min from the edge of a planar glass microchipwith a closed separation channel 10 μm deep, 60 μm wide and 33 mm inlength using electroosmotic flow and applying 4.8 kV to the fluidexiting the closed separation channel on the edge of the microchip forelectrospray formation, with the edge of the chip at a distance of 3-5mm from the ion-sampling orifice of an API mass spectrometer.Approximately 12 nL of the sample fluid collects at the edge of the chipbefore the formation of a Taylor cone and stable nanoelectrospray fromthe edge of the microchip. However, collection of approximately 12 nL ofthe sample fluid will result in remixing of the fluid, thereby undoingthe separation done in the separation channel. Remixing causes bandbroadening at the edge of the microchip, fundamentally limiting itsapplicability for nanoelectrospray-mass spectrometry for analytedetection. Thus, nanoelectrospray from the edge of this microchip deviceafter capillary electrophoresis or capillary electrochromatographyseparation is rendered impractical. Furthermore, because this deviceprovides a flat surface, and thus a relatively small amount of physicalasperity, for the formation of the electrospray, the device requires animpractically high voltage to initiate electrospray, due to poor fieldline concentration.

Xue, Q.; Foret, F.; Dunayevskiy, Y. M.; Zavracky, P. M.; McGruer, N. E.;Karger, B. L. Anal. Chem. 1997, 69, 426-430 describes a stablenanoelectrospray from the edge of a planar glass microchip with a closedchannel 25 μm deep, 60 μm wide and 35-50 mm in length and applying 4.2kV to the fluid exiting the closed separation channel on the edge of themicrochip for electrospray formation, with the edge of the chip at adistance of 3-8 mm from the ion-sampling orifice of an API massspectrometer. A syringe pump is utilized to deliver the sample fluid tothe glass microchip electrosprayer at a flow rate between 100-200nL/min. The edge of the glass microchip is treated with a hydrophobiccoating to alleviate some of the difficulties associated withnanoelectrospray from a flat surface and which slightly improves thestability of the nanoelectrospray. Electrospraying in this manner from aflat surface again results in poor field line concentration and yieldsan inefficient electrospray.

Desai et al. 1997 International Conference on Solid-State Sensors andActuator, Chicago, Jun. 16-19, 1997, 927-930 describes a multi-stepprocess to generate a nozzle on the edge of a silicon microchip 1-3 μmin diameter or width and 40 μm in length and applying 4 kV to the entiremicrochip at a distance of 0.25-0.4 mm from the ion-sampling orifice ofan API mass spectrometer. This nanoelectrospray nozzle reduces the deadvolume of the sample fluid. However, the extension of the nozzle fromthe edge of the microchip exposes the nozzle to accidental breakage.Because a relatively high spray voltage was utilized and the nozzle waspositioned in very close proximity to the mass spectrometer samplingorifice, a poor field line concentration and a low efficientelectrospray were achieved.

In all of the above-described devices, edge-spraying from a microchip isa poorly controlled process due to the inability to rigorously andrepeatedly determine the physical form of the chip's edge. In anotherembodiment of edge-spraying, ejection nozzles, such as small segments ofdrawn capillaries, are separately and individually attached to thechip's edge. This process is inherently cost-inefficient and unreliable,imposes space constraints in chip design, and is therefore unsuitablefor manufacturing.

SUMMARY OF THE INVENTION

The present invention provides a silicon microchip-based electrospraydevice for producing reproducible, controllable and robustnanoelectrospray ionization of a liquid sample. The electrospray devicemay be interfaced downstream to an atmospheric pressure ionization massspectrometer (API-MS) for analysis of the electrosprayed fluid and/orinterfaced upstream to a miniaturized liquid phase separation device,which may have, for example, glass, plastic or silicon substrates orwafers.

The electrospray device of the present invention generally comprises asilicon substrate or microchip defining a channel between an entranceorifice on an injection surface and a nozzle on an ejection surface (themajor surface) such that the electrospray generated by the electrospraydevice is generally approximately perpendicular to the ejection surface.The nozzle has an inner and an outer diameter and is defined by anannular portion recessed from the ejection surface. The annular recessextends radially from the outer diameter. The tip of the nozzle isco-planar or level with and does not extend beyond the ejection surfaceand thus the nozzle is protected against accidental breakage. Thenozzle, channel and recessed portion are etched from the siliconsubstrate by reactive-ion etching and other standard semiconductorprocessing techniques.

All surfaces of the silicon substrate preferably have a layer of silicondioxide thereon created by oxidization to electrically isolate theliquid sample from the substrate and the ejection and injection surfacesfrom each other such that different potential voltages may beindividually applied to each surface and the liquid sample. The silicondioxide layer also provides for biocompatibility. The electrosprayapparatus further comprises at least one controlling electrodeelectrically contacting the substrate through the oxide layer for theapplication of an electric potential to the substrate.

Preferably, the nozzle, channel and recess are etched from the siliconsubstrate by reactive-ion etching and other standard semiconductorprocessing techniques. The injection-side feature(s), through-substratefluid channel, ejection-side features, and controlling electrodes—areformed monolithically from a monocrystalline silicon substrate. That is,they are formed during the course of and as a result of a fabricationsequence that requires no manipulation or assembly of separatecomponents.

Because the electrospray device is manufactured using reactive-ionetching and other standard semiconductor processing techniques, thedimensions of such a device can be very small, for example, as small as2 μm inner diameter and 5 μm outer diameter. Thus, a nozzle having, forexample, 5 μm inner diameter and 250 μm in height only has a volume of4.9 pL (picoliter). In contrast, an electrospray device from the flatedge of a glass microchip would introduce additional dead volume of 12nL compared to the volume of a separation channel of 19.8 nL therebyallowing remixing of the fluid components and undoing the separationdone by the separation channel. The micrometer-scale dimensions of theelectrospray device minimizes the dead volume and thereby increasesefficiency and analysis sensitivity.

The electrospray device of the present invention provides for theefficient and effective formation of an electrospray. By providing anelectrospray surface from which the fluid is ejected with dimensions onthe order of micrometers, the electrospray device limits the voltagerequired to generate a Taylor cone as the voltage is dependent upon thenozzle diameter, surface tension of the fluid and the distance of thenozzle from the extracting electrode. The nozzle of the electrospraydevice provides the physical asperity on the order of micrometers onwhich a large electric field is concentrated. Further, the electrospraydevice may provide additional electrode(s) on the ejecting surface towhich electric potential(s) may be applied and controlled independent ofthe electric potentials of the fluid and the extracting electrode inorder to advantageously modify and optimize the electric field. Thecombination of the nozzle and the additional electrode(s) thus enhancethe electric field between the nozzle and the extracting electrode. Thelarge electric field, on the order of 10₆ V/m or greater and generatedby the potential difference between the fluid and extracting electrode,is thus applied directly to the fluidic cone rather than uniformlydistributed in space.

The microchip-based electrospray ionization device of the presentinvention provides minimal extra-column dispersion as a result of areduction in the extra-column volume and provides efficient,reproducible, reliable and rugged formation of an electrospray. Thedesign of the ionization device is also robust such that theelectrospray device can be readily mass-produced in a cost-effective,high-yielding process.

In operation, a conductive or partly conductive liquid sample isintroduced into the channel through the entrance orifice on theinjection surface. The liquid sample and nozzle are held at thepotential voltage applied to the fluid, either by means of a wire withinthe fluid delivery channel to the electrospray device or by means of anelectrode formed on the injection surface isolated from the surroundingsurface region and from the substrate. The electric field strength atthe tip of the nozzle is enhanced by the application of a voltage to thesubstrate and/or the ejection surface, preferably approximately lessthan one-half of the voltage applied to the fluid. Thus, by theindependent control of the fluid/nozzle and substrate/ejection surfacevoltages, the electrospray device of the present invention allows theoptimization of the electric field lines emanating from the nozzle.Further, when the electrospray device is interfaced downstream with amass spectrometry device, the independent control of the fluid/nozzleand substrate/ejection surface voltages also allows for the directionand optimization of the electrospray into an acceptance region of themass spectrometry device.

The electrospray device of the present invention may be placed 1-2 mm orup to 10 mm from the orifice of an API mass spectrometer to establish astable nanoelectrospray at flow rates as low as 20 nL/min with a voltageof, forexample, 700 V applied to the nozzle and 0-350 V applied to thesubstrate and/or the planar ejection surface of the silicon microchip.

An array or matrix of multiple electrospray devices of the presentinvention may be manufactured on a single microchip as siliconfabrication using standard, well-controlled thin-film processes not onlyeliminates handling of such micro components but also allows for rapidparallel processing of functionally alike elements. The nozzles may beradially positioned about a circle having a relatively small diameternear the center of the chip. Thus, the electrospray device of thepresent invention provides significant advantages of time and costefficiency, control, and reproducibility. The low cost of theseelectrospray devices allows for one-time use such thatcross-contamination from different liquid samples may be eliminated.

The electrospray device of the present invention can be integratedupstream with miniaturized liquid sample handling devices and integrateddownstream with an API mass spectrometer. The electrospray device may bechip-to-chip or wafer-to-wafer bonded to silicon microchip-based liquidseparation devices capable of, for example, capillary electrophoresis,capillary electrochromatography, affinity chromatography, liquidchromatography (LC) or any other condensed-phase separation technique.The electrospray device may be alternatively bonded to glass-and/orpolymer-based liquid separation devices with any suitable method.

In another aspect of the invention, a microchip-based liquidchromatography device may be provided. The liquid chromatography devicegenerally comprises a separation substrate or wafer defining anintroduction channel between an entrance orifice and a reservoir and aseparation channel between the reservoir and an exit orifice. Theseparation channel is populated with separation posts extending from aside wall of the separation channel perpendicular to the fluid flowthough the separation channel. Preferably, the separation posts do notextend beyond and are preferably coplanar or level with the surface ofthe separation substrate such that they are protected against accidentalbreakage during the manufacturing process. Component separation occursin the separation channel where the separation posts perform the liquidchromatography function by providing large surface areas for theinteraction of fluid flowing through the separation channel. A coversubstrate may be bonded to the separation substrate to enclose thereservoir and the separation channel adjacent the cover substrate.

The liquid chromatography device may further comprise one or moreelectrodes for application of electric potentials to the:fluid atlocations along the fluid path. The application of different electricpotentials along the fluid path may facilitate the fluid flow throughthe fluid path.

The introduction and separation channels, the entrance and exit orificesand the separation posts are preferably etched from a silicon substrateby reactive-ion etching and other standard semiconductor processingtechniques. The separation posts are preferably oxidized silicon postswhich may be chemically modified to optimize the interaction of thecomponents of the sample fluid with the stationary separation posts.

In another aspect of the invention, the liquid chromatography device maybe integrated with the electrospray device such that the exit orifice ofthe liquid chromatography device forms a homogenous interface with theentrance orifice of the electrospray device, thereby allowing theon-chip delivery of fluid from the liquid chromatography device to theelectrospray device to generate an electrospray. The nozzle, channel andrecessed portion of the electrospray device may be etched from the coversubstrate of the liquid chromatography device.

In yet another aspect of the invention, multiples of the liquidchromatography-electrospray system may be formed on a single chip todeliver a multiplicity of samples to a common point for subsequentsequential analysis. The multiple nozzles of the electrospray devicesmay be radially positioned about a circle having a relatively smalldiameter near the center of the single chip.

The radially distributed array of electrospray nozzles on a multi-systemchip may be interfaced with a sampling orifice of a mass spectrometer bypositioning the nozzles near the sample orifice. The tight radialconfiguration of the electrospray nozzles allows the positioning thereofin close proximity to the sampling orifice of a mass spectrometer.

The multi-system chip thus provides a rapid sequential chemical analysissystem fabricated using microelectromechanical systems (MEMS)technology. For example, the multi-system chip enables automated,sequential separation and injection of a multiplicity of samples,resulting in significantly greater analysis throughput and utilizationof the mass spectrometer instrument for, for example, high-throughputdetection of compounds for drug discovery.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

FIG. 1 shows a schematic of an electrospray system;

FIG. 2 shows a perspective view of an electrospray device of the presentinvention;

FIG. 3 shows a plan view of the electrospray device of FIG. 2;

FIG. 4 shows a cross-sectional view of the electrospray device of FIG. 3taken along line 4-4;

FIG. 5 shows a schematic of an electrospray system comprising anelectrospray device of the present invention;

FIG. 6 shows a plan view of an electrospray device having multipleelectrodes on the ejection surface of the device;

FIG. 7 shows a cross-sectional view of the electrospray device of FIG. 6taken along line 7-7;

FIG. 8 illustrates a feedback control circuit incorporating anelectrospray device of the present invention;

FIGS. 9-20G show an example of a fabrication sequence of theelectrospray device;

FIG. 21A shows a cross-sectional view of a piezoelectric pipettepositioned at a distance from and for delivery of a fluid sample to theentrance orifice of the electrospray device;

FIG. 21B shows a cross-sectional view of a capillary for delivery of afluid sample to and prior to attachment to the entrance orifice of theelectrospray device;

FIG. 22 shows a schematic of a single integrated system comprising anupstream fluid delivery device and an electrospray device having ahomogeneous interface with the fluid delivery device;

FIG. 23A shows an exploded perspective view of a chip-basedcombinatorial chemistry system comprising a reaction well block and adaughter plate;

FIG. 23B shows a cross-sectional view of the chip-based combinatorialchemistry system of FIG. 23A taken along line 23B-23B;

FIGS. 24A and 24B shows a real Taylor cone emanating from an integratedsilicon chip-based nozzle;

FIGS. 24C and 24D are perspective and side cross-sectional views,respectively, of the electrospray device and mass spectrometry system ofFIGS. 24A and 24B;

FIG. 24E shows a mass spectrum of 1 μg/mL PPG425 in 50% water, 50%methanol containing 0.1% formic acid, 0.1% acetonitrile and 2 mMammonium acetate, collected at a flow rate of 333 nL/min;

FIG. 25A shows an exploded perspective view of a liquid chromatographydevice for homogeneous integration with the electrospray device of thepresent invention;

FIG. 25B shows a cross-sectional view of the liquid chromatographydevice of FIG. 25A taken along line 25B-25B;

FIG. 26 shows a plan view of a liquid chromatography device having anexit orifice forming an off-chip interconnection with an off-chipdevice;

FIG. 27 shows a plan view of a liquid chromatography device having anexit orifice forming an on-chip interconnection with another on-chipdevice;

FIGS. 28-29 show cross-sectional views of liquid chromatography deviceshaving alternative configurations;

FIGS. 30-35 show plan views of liquid chromatography devices havingalternative configurations;

FIGS. 36A-46C show an example of a fabrication sequence of the liquidchromatography device;

FIG. 47 shows a cross-sectional view of a system comprising a liquidchromatography device homogenously integrated with an electrospraydevice;

FIG. 48 shows a plan view of the system of FIG. 47; and

FIG. 49 shows a detailed view of the nozzles of the system of FIG. 47.

DETAILED DESCRIPTION OF THE INVENTION

An aspect of the present invention provides a silicon microchip-basedelectrospray device for producing electrospray ionization of a liquidsample. The electrospray device may be interfaced downstream to anatmospheric pressure ionization mass spectrometer (APIMS) for analysisof the electrosprayed fluid. Another aspect of the invention is anintegrated miniaturized liquid phase separation device, which may have,for example, glass, plastic or silicon substrates integral with theelectrospray device. The descriptions that follow present the inventionin the context of a liquid chromatograph separation device. However itwill be readily recognized that equivalent devices can be made thatutilize other microchip-based separation devices. The followingdescription is presented to enable any person skilled in the art to makeand use the invention. Descriptions of specific applications areprovided only as examples. Various modifications to the preferredembodiment will be readily apparent to those skilled in the art, and thegeneral principles defined herein may be applied to other embodimentsand applications without departing from the spirit and scope of theinvention. Thus, the present invention is not intended to be limited tothe embodiments shown, but is to be accorded the widest scope consistentwith the principles and features disclosed herein.

Electrospray Device

FIGS. 2-4 show, respectively, a perspective view, a plan view and acrosssectional view of an electrospray device 100 of the presentinvention. The electrospray apparatus of the present invention generallycomprises a silicon substrate or microchip or wafer 102 defining achannel 104 through substrate 102 between an entrance orifice 106 on aninjection surface 108 and a nozzle 110 on an ejection surface 112. Thechannel may have any suitable cross-sectional shape such as circular orrectangular. The nozzle 110 has an inner and an outer diameter and isdefined by a recessed region 114. The region 114 is recessed from theejection surface 112, extends outwardly from the nozzle 110 and may beannular. The tip of the nozzle 110 does not extend beyond and ispreferably coplanar or level with the ejection surface 112 to therebyprotect the nozzle 110 from accidental breakage.

Preferably, the injection surface 108 is opposite the ejection surface112. However, although not shown, the injection surface may be adjacentto the ejection surface such that the channel extending between theentrance orifice and the nozzle makes a turn within the device. In sucha configuration, the electrospray device would comprise two substratesbonded together. The first substrate may define a through-substratechannel extending between a bonding surface and the ejection surface,opposite the bonding surface. The first substrate may further define anopen channel recessed from the bonding surface extending from an orificeof the through-substrate channel and the injection surface such that thebonding surface of the second substrate encloses the open channel uponbonding of the first and second substrates. Alternatively, the secondsubstrate may define an open channel recessed from the bonding surfacesuch that the bonding surface of the first substrate encloses the openchannel upon bonding of the first and second substrates. In yet anothervariation, the first substrate may further define a secondthrough-substrate channel while the open channel extends between the twothrough-substrate channels. Thus, the injection surface is the samesurface as the ejection surface.

A grid-plane region 116 of the ejection surface 112 is exterior to thenozzle 110 and to the recessed region 114 and may provide a surface onwhich a layer of conductive material 119, including a conductiveelectrode 120, may be formed for the application of an electricpotential to the substrate 102 to modify the electric field patternbetween the ejection surface 112, including the nozzle tip 110, and theextracting electrode 54. Alternatively, the conductive electrode may beprovided on the injection surface 108 (not shown).

The electrospray device 100 further comprises a layer of silicon dioxide118 over the surfaces of the substrate 102 through which the electrode120 is in contact with the substrate 102 either on the ejection surface112 or on the injection surface 108. The silicon dioxide 118 formed onthe walls of the channel 104 electrically isolates a fluid therein fromthe silicon substrate 102 and thus allows for the independentapplication and sustenance of different electrical potentials to thefluid in the channel 104 and to the silicon substrate 102. The abilityto independently vary the fluid and substrate potentials allows theoptimization of the electrospray through modification of the electricfield line pattern, as described below. Alternatively, the substrate 102can be controlled to the same electrical potential as the fluid whenappropriate for a given application.

As shown in FIG. 5, to generate an electrospray, fluid may be deliveredto the entrance orifice 106 of the electrospray device 100 by, forexample, a capillary 52 or micropipette. The fluid is subjected to apotential voltage V_(fluid) via a wire (not shown) positioned in thecapillary 52 or in the channel 104 or via an electrode (not shown)provided on the injection surface 108 and isolated from the surroundingsurface region and the substrate 102 . A potential voltage V_(substrate)may also be applied to the electrode 120 on the grid-plane 116, themagnitude of which is preferably adjustable for optimization of theelectrospray characteristics. The fluid flows through the channel 104and exits or is ejected from the nozzle 110 in the form of very fine,highly charged fluidic droplets 58. The electrode 54 may be held at apotential voltage V_(extract) such that the electrospray is drawn towardthe extracting electrode 54 under the influence of an electric field. Asit is the relative electric potentials which affect the electric field,the potential voltages of the fluid, the substrate and the extractingelectrode may be easily adjusted and modified to achieve the desiredelectric field. Generally, the magnitude of the electric field shouldnot exceed the dielectric breakdown strength of the surrounding medium,typically air.

In one embodiment, the nozzle 110 may be placed up to 10 mm from thesampling orifice of an API mass spectrometer serving as the extractingelectrode 54. A potential voltage V_(fluid) ranging from approximately500-1000 V, such as 700 V, is applied to the fluid. The potentialvoltage of the fluid V_(fluid) may be up to 500 V/μm of silicon dioxideon the surface of the substrate 102 and may depend on the surfacetension of the fluid being sprayed and the geometry of the nozzle 110. Apotential voltage of the substrate V_(substrate) of approximately lessthan half of the fluid potential voltage V_(fluid), or 0-350 V, isapplied to the electrode on the grid-plane 116 to enhance the electricfield strength at the tip of the nozzle 110. The extracting electrode 54may be held at or near ground potential V_(extract) (0 V). Thus, ananoelectrospray of a fluid introduced to the electrospray device 100 atflow rates less than 1,000 nL/min is drawn toward the extractingelectrode 54 under the influence of the electric field.

The nozzle 110 provides the physical asperity for concentrating theelectric field lines emanating from the nozzle 110 in order to achieveefficient electrospray. The nozzle 110 also forms a continuation of andserves as an exit orifice of the through-substrate channel 104.Furthermore, the recessed region 114 serves to physically isolate thenozzle 110 from the grid-plane region 116 of the ejection surface 112 tothereby promote the concentration of electric field lines and to provideelectrical isolation between the nozzle 110 and the grid-plane region116. The present invention allows the optimization of the electric fieldlines emanating from the nozzle 110 through independent control of thepotential voltage V_(fluid) of the fluid and nozzle 110 and thepotential voltage V_(substrate) of the electrode on the grid-plane 116of the ejection surface 112.

In addition to the electrode 120, one or more additional conductiveelectrodes may be provided on the silicon dioxide layer 118 on theejection surface 112 of the substrate 102. FIGS. 6 and 7 show,respectively, a plan view and a cross-sectional view of an example of anelectrospray device 100′ wherein the conductive layer 119 defines threeadditional electrodes 122, 124, 126 on the ejection surface 112 of thesubstrate 102. Because the silicon dioxide layer 118 on the ejectionsurface 112 electrically isolates the silicon substrate 102 from theadditional electrodes 122, 124, 126 on the ejection surface 112 andbecause the additional electrodes 122, 124, 126 are physically separatedfrom each other, the electrical potential applied to each of theadditional electrodes 122, 124, 126 can be controlled independently fromeach other, from the substrate 102 and from the fluid. Thus, additionalelectrodes 122, 124, 126 may be utilized to further modify the electricfield line pattern to effect, for example, a steering and/or shaping ofthe electrospray. Although shown to be of similar sizes and shapes,electrode 120 and additional electrodes 122, 124, 126 may be of any sameor different suitable shapes and sizes.

To further control and optimize the electrospray, a feedback controlcircuit 130 as shown in FIG. 8 may also be provided with theelectrospray device 100. The feedback circuit 130 includes an optimalspray attribute set point 132, a comparator and voltage control 134 andone or more spray attribute sensors 136. The optimal spray attribute setpoint 132 is set by an operator or at a determined or default value. Theone or more spray attribute sensors 136 detect one or more desiredattributes of the electrospray from the electrospray device 100, such asthe electrospray ion current and/or the spatial concentration of thespray pattern. The spray attribute sensor 136 sends signals indicatingthe value of the desired attribute of the electrospray to the comparatorand voltage control 134 which compares the indicated value of thedesired attribute with the optimal spray attribute set point 132. Thecomparator and voltage control 134 then applies: potential voltagesV_(fluid), V_(substrate) to the fluid and the silicon substrate 102,respectively, which may be independently varied to optimize the desiredelectrospray attribute. Although not shown, the comparator and voltagecontrol 134 may apply independently controlled additional potentialvoltages to each of one or more additional conductive electrodes.

The feedback circuit 130 may be interfaced with the electrospray device100 in any suitable fashion. For example, the feedback circuit 130 maybe fabricated as an integrated circuit on the electrospray device 100,as a separate integrated circuit with electrical connection to theelectrospray device 100, or as discrete components residing on a commonsubstrate electrically connected to the substrate of the electrospraydevice.

Dimensions of the electrospray device 100 can be determined according tovarious factors such as the specific application, the layout design aswell as the upstream and/or downstream device to which the electrospraydevice 100 is interfaced or integrated. Further, the dimensions of thechannel and nozzle may be optimized for the desired flow rate of thefluid sample. The use of reactive-ion etching techniques allows for thereproducible and cost effective production of small diameter nozzles,for example, a 2 μm inner diameter and 5 μm outer diameter.

In one currently preferred embodiment, the silicon substrate 102 of theelectrospray device 100 is approximately 250-600 μm in thickness and thecross-sectional area of the channel 104 is less than approximately50,000 μm². Where the channel 104 has a circular cross-sectional shape,the channel 104 and the nozzle 110 have an inner diameter of up to 250μm, more preferably up to 145 μm; the nozzle 110 has an outer diameterof up to 255 μm, more preferably up to 150 μm; and nozzle 110 has aheight of (and the recessed portion 114 has a depth of) up to 500 μm.The recessed portion 114 preferably extends up to 1000 m outwardly fromthe nozzle 110. The silicon dioxide layer 118 has a thickness ofapproximately 1-4 μm, preferably 1-2 μm.

Electrospray Device Fabrication Procedure

The fabrication of the electros pray device 100 will now be explainedwith reference to FIGS. 9-20B. The electrospray device 100 is preferablyfabricated as a monolithic silicon integrated circuit utilizingestablished, well-controlled thin-film silicon processing techniquessuch as thermal oxidation, photolithography, reactivation etching (RIE),ion implantation, and metal deposition. Fabrication using such siliconprocessing techniques facilitates massively parallel processing ofsimilar devices, is time- and cost-efficient, allows for tighter controlof critical dimensions, is easily reproducible, and results in a whollyintegral device, thereby eliminating any assembly requirements. Further,the fabrication sequence may be easily extended to create physicalaspects or features on the injection surface and/or ejection surface ofthe electrospray device to facilitate interfacing and connection to afluid delivery system or to facilitate integration with a fluid deliverysub-system to create a single integrated system.

Injection Surface Processing: Entrance to Through-wafer Channel

FIGS. 9A-11 illustrate the processing steps for the injection side ofthe substrate in fabricating the electrospray device 100 of the presentinvention. Referring to the plan and cross-sectional views,respectively, of FIGS. 9A and 9B, a double-side polished silicon wafersubstrate 200 is subjected to an elevated temperature in an oxidizingambient to grow a layer or film of silicon dioxide 202 on the injectionside 203 and a layer or film of silicon dioxide 204 on the ejection side205 of the substrate 200. Each of the resulting silicon dioxide layers202, 204 has a thickness of approximhately 1-2 μm. The silicon dioxidelayers 202, 204 provide electrical isolation and also serve as masks forsubsequent selective etching of certain areas of the silicon substrate200.

A film of positive-working photoresist 206 is deposited on the silicondioxide layer 202 on the injection side 203 of the substrate 200. Anarea of the photoresist 206 corresponding to the entrance to athrough-wafer channel which will be subsequently etched is selectivelyexposed through a mask by an optical lithographic exposure tool passingshort-wavelength light such as blue or near-ultraviolet at wavelengthsof 365, 405, or 436 nanometers.

As shown in the plan and cross-sectional views respectively, of FIGS.10A and 10B, after development of the photoresist 206, the exposed area208 of the photoresist is removed and open to the underlying silicondioxide layer 202 while the unexposed areas remain protected byphotoresist 206′. The exposed area 210 of the silicon dioxide layer 202is then etched by a fluorine-based plasma with a high degree ofanisotropy and selectivity to the protective photoresist 206′ until thesilicon substrate 200 is reached. The remaining photoresist is removedin an oxygen plasma or in an actively oxidizing chemical bath likesulfuric acid (H₂SO₄) activated with hydrogen peroxide (H₂O₂).

As shown in the cross-sectional view of FIG. 1, an injection sideportion 212 of the through channel in the silicon substrate 200 isvertically etched by another fluorine-based etch. An advantage of thefabrication process described herein is that the dimensions of thethrough channel, such as the aspect ratio (depth to width), can bereliably and reproducibly limited and controlled. In the case where theetch aspect ratio of the processing equipment is a limiting factor, itis possible to overcome this limitation by a first etch on one side of awafer followed by a second etch on a second side of the wafer. Forexample, a current silicon etch process is generally limited to an etchaspect ratio of 30:1, such that a channel having a diameter less thanapproximately 10 μm through a substrate 200 having customary knessapproximately 250-600 μmm would be etched from both surfaces of thesubstrate 200.

The depth of the channel portion 212 should be at or above a minimum inorder to connect with another portion of the through channel etched fromthe ejection side 205 of the substrate 200. The desired depth of therecessed region 114 on the ejection side 205 determines approximatelyhow far the ejection side portion 220 of the channel 104 is etched. Theremainder of the channel 104, the injection side portion 212, is etchedfrom the injection side. The minimum depth of channel portion 212 istypically 50 μm, although the exact etch depth above the minimum etchdepth does not impact the device performance or yield of theelectrospray device.

Ejection Surface Processing: Nozzle and Surrounding Surface Structure

FIGS. 12-20B illustrate the processing steps for the ejection side 205of the substrate 200 in fabricating the electrospray device 100 of thepresent invention. As shown in the cross-sectional view in FIG. 12, afilm of positive-working photoresist 214 is deposited on the silicondioxide layer 204 on the ejection side 205 of the substrate 200.Patterns on the ejection side 205 are aligned to those previously formedon the injection side 203 of the substrate 200. Because silicon and itsoxide are inherently relatively transparent to light in the infraredwavelength range of the spectrum, i.e. approximately 70-1000 nanometers,the extant pattern on the injection side 203 can be distinguished withsufficient clarity by illuminating the substrate 200 from the patternedinjection side 203 with infrared light. Thus, the mask for the ejectionside 205 can be aligned within required tolerances.

After alignment, certain areas of the photoresist 214 corresponding tothe nozzle and the recessed region are selectively exposed through anejection side mask by an optical lithographic exposure tool passingshort-wavelength light, such as blue or near-ultraviolet at wavelengthsof 365, 405, or 436 nanometers. A s shown in the plan andcross-sectional views, respectively, of FIGS. 13A and 13B, thephotoresist 214 is then developed to remove the exposed areas of thephoto resist such that the nozzle area 216 and recessed region area 218are open to the underlying silicon dioxide layer 204 while the unexposedareas remain protected by photoresist 214′. The exposed areas 216, 218of the silicon dioxide layer 204 are then etched by a fluorine-basedplasma with a high degree of anisotropy and selectivity to theprotective photoresist 214′ until the silicon substrate 200 is reached.

As shown in the cross-sectional view of FIG. 14, the remainingphotoresist 214′ provides additional masking during a subsequentfluorine based silicon etch to vertically etch certain patterns into theejection side 205 of the silicon substrate 200. The remainingphotoresist 214′ is then removed in an oxygen plasma or in an activelyoxidizing chemical bath like sulfuric acid (H₂SO₄) activated withhydrogen peroxide (H₂O₂).

The fluorine-based etch creates a channel 104 through the siliconsubstrate 200 by forming an ejection side portion 220 of the channel104. The fluorine based etch also creates an ejection nozzle 110, arecessed region 114 exterior to the nozzle 110 and a grid-plane region116 exterior to the nozzle 110 and to the recessed region 114. Thegrid-plane region 116 is preferably co-planar with the tip of the nozzle110 so as to physically protect the nozzle 110 from casual abrasion,stress fracture in handling and/or accidental breakage. The grid-planeregion 116 also serves as a platform on which one or more conductiveelectrodes may be provided.

The fabrication sequence confers superior mechanical stability to thefabricated electrospray device by etching the features of theelectrospray device from a monocrystalline silicon substrate without anyneed for assembly. The fabrication sequence allows for the control ofthe nozzle height by adjusting the relative amounts of injection sideand ejection side silicon etching. Further, the lateral extent and shapeof the recessed region 114 can be controlled independently of its depth,which affects the nozzle height and which is determined by the extent ofthe etch on the ejection side of the substrate. Control of the lateralextent and shape of the recessed region 114 provides the ability tomodify and control the electric field pattern between the electrospraydevice 100 and an extracting electrode.

Oxidation for Electrical Isolation

As shown in the cross-sectional view of FIG. 15, a layer of silicondioxide 221 is grown on all silicon surfaces of the substrate 200 bysubjecting the silicon substrate 200 to elevated temperature in anoxidizing ambient. For example, the oxidizing ambient may be anultra-pure steam produced by oxidation of hydrogen for a silicon dioxidethickness greater than approximately several hundred nanometers or pureoxygen for a silicon dioxide thickness of approximately several hundrednanometers or less. The layer of silicon dioxide 221 over all siliconsurfaces of the substrate 200 electrically isolates a fluid in thechannel from the silicon substrate 200 and permits the application andsustenance of different electrical potentials to the fluid in thechannel 104 and to the silicon substrate 200.

All silicon surfaces are oxidized to form silicon dioxide with athickness that is controllable through choice of temperature and time ofoxidation. The final thickness of the silicon dioxide can be selected toprovide the desired degree of electrical isolation in the device, wherea thicker layer of silicon dioxide provides a greater resistance toelectrical breakdown.

Metallization for Electric Field Control

FIGS. 16-20B illustrate the formation of a single conductive electrodeelectrically connected to the substrate 200 on the ejection side 205 ofthe substrate 200. As shown in the cross-sectional view of FIG. 16, afilm of positive-working photoresist 222 is deposited over the silicondioxide layer on the ejection side 205 of the substrate 200. An area ofthe photoresist 222 corresponding to the electrical contact area betweenthe electrode and the substrate 200 is selectively exposed throughanother mask by an optical lithographic exposure tool passingshort-wavelength light, such as blue or near-ultraviolet at wavelengthsof 365, 405, or 436 nanometers.

The photoresist 222 is then developed to remove the exposed area 224 ofthe photoresist such that the electrical contact area between theelectrode and the substrate 200 is open to the underlying silicondioxide layer 204 while the unexposed areas remain protected byphotoresist 222′. The exposed area 224 of the silicon dioxide layer 204is then etched by a fluorine-based plasma with a high degree ofanisotropy and selectivity to the protective photoresist 222′ until thesilicon substrate 200 is reached, as shown in the cross-sectional viewof FIG. 17.

Referring now to the cross-sectional view of FIG. 18, the remainingphotoresist is then removed in an oxygen plasma or in an activelyoxidizing chemical bath like sulfuric acid (H₂SO₄) activated withhydrogen peroxide (H₂O₂). Utilizing the patterned ejection side silicondioxide layer 204 as a mask a high-dose implantation is made to form animplanted region 225 to ensure a low-resistance electrical connectionbetween the electrode and the substrate 200. A conductive film 226 suchas aluminum may be uniformly deposited on the ejection side 205 of thesubstrate 200 by thermal or election-beam evaporation to form anelectrode 120. The thickness of the conductive film 226 is preferablyapproximately 3000 Å, although shown having a larger thickness forclarity.

The conductive film 226 may be created by any method which does notproduce a continuous film of the conductive material on the side wallsof the ejection nozzle 110. Such a continuous film would electricallyconnect the fluid in the channel 104 and the substrate 200 so as toprevent the independent control of their respective electricalpotentials. For example, the conductive film may be deposited by thermalor electron-beam evaporation of the conductive material, resulting inline-of-sight deposition on presented surfaces. Orienting the substrate200 such that the side walls of the ejection nozzle 110 are out of theline-of-sight of the evaporation source ensures that no conductivematerial is deposited as a continuous film on the side walls of theejection nozzle 110. Sputtering of conductive material in a plasma is anexample of a deposition technique which would result in deposition ofconductive material on all surfaces and thus is undesirable.

One or more additional conductive electrodes may be easily formed on theejection side 205 of the substrate 200, as described above withreference to FIGS. 6 and 7. As shown in the cross-sectional view of FIG.19, a film of positive-working photoresist 228 is deposited over theconductive film 226 on the ejection side 205 of the substrate 200.Certain areas of the photoresist 228 corresponding to the physicalspaces between the electrodes are selectively exposed through anothermask by an optical lithographic exposure tool passing short-wavelengthlight, such as blue or near-ultraviolet at wavelengths of 365, 405, or436 nanometers.

Referring now to the plan and cross-sectional views of FIGS. 20A and20B, the photoresist 228 is developed to remove the exposed areas 230 ofthe photoresist such that the exposed areas are open to the underlyingconductive film 226 while the unexposed areas remain protected byphotoresist 228′. The exposed areas 230 of the conductive film 226 arethen etched using either a wet chemical etch or a reactive-ion etch, asappropriate for the particular conductive material. The etch is eitherselective to the underlying silicon dioxide layer 204 or the etch mustbe terminated on the basis of etch rate and time of etch. Finally, theremaining photoresist is then removed in an oxygen plasma.

The etching of the conductive film 226 to the underlying silicon dioxidelayer 204 results in physically and electrically separate islands ofconductive material or electrodes. As described above, these electrodescan be controlled independently from the silicon substrate or channelfluid because they are electrically isolated from the substrate by thesilicon dioxide and from each other by physical separation. They can beused to further modify the electric field line pattern and therebyeffect a steering and/or shaping of the electrosprayed fluid. This stepcompletes the processing and fabrication sequence for the electrospraydevice 100.

As described above, the conductive electrode for application of anelectrical potential to the substrate of the electrospray device may beprovided on the injection surface rather than the ejection surface. Thefabrication sequence is similar to that for the conductive electrodeprovided on the ejection side 205 of the substrate 200. FIGS. 20C-20Gillustrate the formation of a single conductive electrode electricallyconnected to the substrate 200 on the injection side 203 of thesubstrate 200.

As shown in the cross-sectional view of FIG. 20C, a film ofpositive-working photoresist 232 is deposited over the silicon dioxidelayer on the injection side 203 of the substrate 200. An area of thephotoresist 232 corresponding to the electrical contact area between theelectrode and the substrate 200 is selectively exposed through anothermask by an optical lithographic exposure tool passing shortwave lengthlight, such as blue or near ultraviolet at wavelengths of 365, 405, or436 nanometers.

The photoresist 232 is then developed to remove the exposed area 234 ofthe photoresist such that the electrical contact area between theelectrode and the substrate 200 is open to the underlying Silicondioxide layer 202 while the unexposed areas remain protected byphotoresist 232′. The exposed area 234 of the silicon dioxide layer 202is then etched by a fluorine-based plasma with a high degree ofanisotropy and selectivity to the protective photoresist 232′ until thesilicon substrate 200 is reached, as shown in the cross-sectional viewof FIG. 20D.

Referring now to the cross-sectional view of FIG. 20E the remainingphotoresist is then removed in an oxygen plasma or in an activelyoxidizing chemical bath like sulfuric acid (H₂SO₄) activated withhydrogen peroxide (H₂O₂). Utilizing the patterned injection side silicondioxide layer 202 as a mask, a: high-dose implantation is made to forman implanted region 236 to ensure a low-resistance electrical connectionbetween the electrode and the substrate 200. A conductive film 238i:such as aluminum may be uniformly deposited on the injection side 203 ofthe substrate 200 by thermal or electron beam evaporation to form anelectrode 120′.

In contrast to the formation of the conductive electrode on the ejectionsurface of the electrospray device, sputtering, in addition to thermalor electron-beam evaporation, may be utilized to form the conductiveelectrode on the injection surface. Because the nozzle is on theejection rather than the injection side of the substrate, sputtering maybe utilized to form the electrode on the injection side as the injectionside electrode layer does not extend to the nozzle to create aphysically continuous and thus electrically conductive path with thenozzle.

With the formation of the electrode on the injection surface of theelectrospray device, sputtering may be preferred over evaporationbecause of its greater ability to produce conformal coatings on thesidewalls of the exposed area 234 etched through the silicon dioxidelayer 202 to the substrate 200 to ensure electrical continuity andreliable electrical contact to the substrate 200.

For certain applications, it may be necessary to ensure electricalisolation between the substrate 200 and the fluid in the electrospraydevice by removing the conductive film from the region of the surfaceadjacent to the entrance orifice 106 on the injection side 203. Theextent of the conductive film 238 which should be removed isirrespective of etching method and may be determined by the specificmethod utilized in creating the interface between the upstream fluiddelivery system/sub-system and the injection side of the electrospraydevice. For example, a diameter of between approximately 0.2-2 mm of theconductive film 238 may be removed from the region surrounding theentrance orifice 106.

As shown in the cross-sectional view of FIG. 20F, another film ofpositive-working photoresist 240 is deposited over the conductive film238 on the injection side 203 of the substrate 200. An area of thephotoresist 240 corresponding to the region adjacent to the entranceorifice 106 on the injection side 203 is selectively exposed throughanother mask by an optical lithographic exposure tool passingshort-wavelength light, such as blue or near-ultraviolet at wavelengthsof 365, 405, or 436 nanometers.

The photoresist 240 is then developed to remove the exposed area 242 ofthe photoresist such that the region adjacent to the entrance orifice106 on the injection side 203 is open to the underlying conductive film238 while the unexposed areas remain protected by photoresist 240′. Theexposed area 242 of the conductive film 238 is then etched by, forexample, a chlorine-based plasma with a high degree of anisotropy andselectivity to the protective photoresist 240′ until the silicon dioxidelayer 203 is reached, as shown in the cross-sectional view of FIG. 20G.

The specific technique for etching the conductive film 238 may bedetermined by the specific conductive material deposited. For example,aluminum may be etched either in a wet chemical bath using standardaluminum etchant or in a plasma using reactive-ion etching (RIE) andchlorine-based gas chemistry. Utilization of standard wet aluminumetchant to etch an aluminum film may be preferred as such wet etchingmay facilitate the removal of any undesired conductive materialdeposited in the channel 104 via the entrance orifice 106. Further,although chlorine-based reactive-ion etching may be utilized, suchetching may lead to aluminum corrosion if removal of the photoresist isdelayed.

Forming the electrode on the injection surface for application of anelectric potential to the substrate of the electrospray device mayprovide several advantages. For example, because the ability touniformly coat photoresist on a surface is limited by nonplanar surfacetopology, coating photoresist on the much flatter injection side resultsin a more uniform and continuous photoresist film than coatingphotoresist on the ejection side. The uniformity and continuity of thephotoresist film directly and positively impact the reliability andyield, at least in part because failure of photoresist coverage wouldallow subsequent etching of silicon dioxide in undesired locationsduring the etching of exposed areas 224, 234.

Another advantage of forming the electrode on the injection surface isthe greater flexibility and reliability in the conductive materialdeposition step because the interior surfaces of the nozzle are notcoated by the conductive material deposited onto the injection surfacerather than onto the ejection surface of the electrospray device. As aresult, sputtering may be utilized as a deposition technique to ensureconformal coating of the conductive material and electrical continuityfrom the surface to the substrate contact. Further, the provision of theelectrode on the injection surface does not preclude the deposition andpatterning of additional conductive electrodes on the ejection side tofurther modify the electric field line pattern to effect, for example, asteering and/or shaping of the electrospray, as such additionalelectrodes do not required electrical contact to the substrate.

The ability to form the electrode on the injection surface may also beadvantageous in certain applications where physical constraints, such asin packaging, may dictate the need for injection-side rather thanejection-side electrical connection.

The above described fabrication sequence for the electrospray device 100can be easily adapted to and is applicable for the simultaneousfabrication of a single monolithic system comprising multipleelectrospray devices including multiple channels and/or multipleejection nozzles embodied in a single monolithic substrate. Further, theprocessing steps may be modified to fabricate similar or differentelectrospray devices merely by, for example, modifying the layout designand/or by changing the polarity of the photomask and utilizingnegative-working photoresist rather than utilizing positive-workingphotoresist.

Further, although the fabrication sequence is described in terms offabricating a single electrospray device, the fabrication sequencefacilitates and allows for massively parallel processing of similardevices. The multiple electrospray devices or systems fabricated bymassively parallel processing on a single wafer may then be cut orotherwise separated into multiple devices or systems.

Interface or Integration of the Electrospray Device

Downstream Interface or Integration of the Electrospray Device

The electrospray device 100 may be interfaced or integrated downstreamto a sampling device, depending on the particular application. Forexample the analyte may be electrosprayed onto a surface to coat thatsurface or into another device for purposes of conveyance, analysis,and/or synthesis. As described above with reference to FIG. 5, highlycharged droplets are formed at atmospheric pressure by the electrospraydevice 100 from nanoliter-sc ale volumes of an analyte. The highlycharged droplets produce gas-phase ions upon sufficient evaporation ofsolvent molecules which may be sampled, for example, through an orificeof an atmospheric pressure ionization mass spectrometer (API-MS) foranalysis of the electrosprayed fluid.

Upstream Interface or Integration of the Electrospray Device

Referring now to FIGS. 21-23, fluid may be delivered to the entranceorifice of the electrospray device in any suitable manner by upstreaminterface or integration with one or more fluid delivery devices, suchas piezoelectric pipettes, micropipettes, capillaries and other types ofmicrodevices. The fluid delivery device may be a separate component toform a heterogeneous interface with the entrance orifice of theelectrospray device. Alternatively, the fluid delivery device may beintegrated with the electrospray device to form a homogeneous interfacewith the entrance orifice of the electrospray device.

FIGS. 21A and 21B illustrate examples of fluid delivery devices formingheterogeneous interfaces with the entrance orifice of the electrospraydevice. Preferably, the heterogeneous interface is a non-contactinginterface where the fluid delivery device and the electrospray deviceare physically separated and do not contact. For example, as shown inthe cross-sectional view of FIG. 21A, a piezoelectric pipette 300 ispositioned at a distance above the injection surface 108 of theelectrospray device 100A. The piezoelectric pipette 300 deposits a flowof microdroplets, each approximately 200 pL in volume, into the channel104 through the entrance orifice 106A. Preferably, the electrospraydevice 100A provides an entrance well 302 at the entrance orifice 106Afor containing the sample fluid prior to entering the channel 104particularly when it is desirable to spray a volume of fluid greaterthan the volume of the through-substrate channel 104 and continualsupply of fluid is not feasible such as when using the piezoelectricpipette 300. The entrance well 302 preferably has a volume of 0.1 nL to100 nL. Furthermore, to apply an electric potential to the fluid, anentrance well electrode 304 may be provided on a surface of the entrancewell 302 parallel to the injection surface 108. Alternatively, a wire(not shown) may be positioned in channel 104 via the entrance orifice106A. Preferably, some fluid is present in the entrance well 302 toensure electrical contact between the fluid and the entrance wellelectrode 304.

Alternatively, the heterogeneous interface may be a contacting interfacewhere a fluid delivery device is attached by any suitable method, suchas by epoxy bonding, to the electrospray device to form a continuoussealed flow path between the upstream fluid source and the channel ofthe electrospray device. For example, FIG. 21B shows a cross-sectionalview of a capillary 306 prior to attachment to the entrance orifice 106of the electrospray device 100B. The injection surface 108 of theelectrospray device 100B may be adapted to facilitate attachment of thecapillary 306. Such features can be easily designed into the mask forthe injection side of the substrate and can be simultaneously formedwith the injection side portion of the channel during the etchingperformed on the injection-side.

For example, where the inner diameter of the capillary 306 is greaterthan that of the channel 104 and the entrance orifice 106, theelectrospray device 100B preferably defines a region 308 recessed fromthe injection surface 108 to form a mating collar for mating andaffixing with the capillary 306. Thus, capillary 306 may be positionedand attached in the recessed region 308 such that the exit orifice 310portion of the capillary 302 is positioned around the entrance orifice106. Further, the electrospray device 100B may optionally provide anentrance well 312 at the entrance orifice 106B for containing the samplefluid prior to entering the channel 104. Although not shown, if theouter diameter of the capillary is less than that of the channel and theentrance orifice, the capillary may be inserted into and attached to theentrance orifice of the electrospray device.

Referring now to the schematic of FIG. 22, rather than a heterogeneousinterface, a single integrated system 316 is provided wherein anupstream fluid delivery device 318 forms a homogeneous interface withthe entrance orifice (not shown) of an electrospray device 100. Thesystem 316 allows for the fluid exiting the upstream fluid deliverydevice 318 to be delivered on-chip to the entrance orifice of theelectrospray device 100 in order to generate an electrospray.

The single integrated system 316 provides the advantage of minimizing oreliminating extra fluid volume to reduce the risk of undesired fluidchanges, such as by reactions and/or mixing. The single integratedsystem 316 also provides the advantage of eliminating the need forunreliable handling and attachment of components at the microscopiclevel and of minimizing or eliminating fluid leakage by containing thefluid within one integrated system.

The upstream fluid delivery device 318 may be a monolithic integratedcircuit having an exit orifice through which a fluid sample can passdirectly or indirectly to the entrance orifice of the electrospraydevice 100. The upstream fluid delivery device 318 may be a siliconmicrochip-based liquid separation device capable of, for example,capillary electrophoresis, capillary electrochromatography, affinitychromatography, liquid chromatography (LC) or any other condensed-phaseseparation methods. Further, the upstream fluid delivery device 318 maybe a silicon, glass, plastic and/or polymer based device such that theelectrospray device 100 may be chip-to-chip or wafer-to-wafer bondedthereto by any suitable method. An example of a monolithic liquidchromatography device for utilization in, for example, the singleintegrated system 316, is described below.

Electrospray Device for Sample Transfer of Combinatorial ChemistryLibraries Synthesized in Microdevices

The electrospray device may also serve to reproducibly distribute anddeposit a sample from a mother plate to daughter plate(s) bynanoelectrospray deposition. Electrospray device(s) may be etched into amicrodevice capable of synthesizing combinatorial chemical libraries. Atthe desired time, the nozzle may spray a desired amount of the samplefrom the mother plate to the daughter plate(s). Control of the nozzledimensions, applied voltages, and time of spraying may provide a preciseand reproducible method of sample deposition from an array of nozzles,such as the generation of sample plates for molecular weightdeterminations by matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOFMS). The capability oftransferring analytes from a mother plate to daughter plates may also beutilized to make other daughter plates for other types of assays, suchas proteomic screening.

FIGS. 23A and 23B show; respectively, an exploded perspective view and across-sectional view along line 23B-23B, of a chip-based combinatorialchemistry system 320 comprising a reaction well block or titer plate 322and a receiving or daughter plate 324. The reaction well block 322defines an array of reservoirs 326 for containing the reaction productsfrom a combinatorially synthesized compound. The reaction well block 322further defines channels 328, nozzles 330 and recessed portions 332 suchthat the fluid in each reservoir 326 may flow through a correspondingchannel 328 and exit through a corresponding nozzle 330 in the form ofan electrospray. The reaction well block 322 may define any number ofreservoir(s) in any desirable configuration, each reservoir being of asuitable dimension and shape. The volume of a reservoir 326 may rangefrom a few nanoliters up to several microliters and more preferablyranges between approximately 200 nL to 1 μL.

The reaction well block 322 may serve as a mother plate to interface toa microchip-based chemical synthesis apparatus such that theelectrospray function of the reaction well block 322 may be utilized toreproducibly distribute discreet quantities of the product solutions toa receiving or daughter plate 324. The daughter plate 324 definesreceiving wells 334 which correspond to each of the reservoirs 326. Thedistributed product solutions in the daughter plate 324 may then beutilized to screen the combinatorial chemical library against biologicaltargets.

Illustration of an Electrospray Device Generating an Electrospray Spray

FIGS. 24A and 24B show color images of a real Taylor cone emanating froman integrated silicon chip-based nozzle. FIGS. 24C and 24D areperspective and side cross-sectional views, respectively, of theelectrospray device and mass spectrometer system shown in FIGS. 24A and24B. FIGS. 24A shows a chip-integrated electrospray device comprising anozzle and a recessed portion or annulus, and a Taylor cone, liquid jetand plume of highly-charged electrosprayed droplets of methanolcontaining 10 μg/mL polypropylene glycol 425 (PPG425) containing 0.2%formic acid. FIG. 24B shows an ion-sampling orifice of a massspectrometer in addition to the electrospray device.

The electrospray device 100 is interfaced upstream with a pipette 52′.As shown in the upper right corner of each of FIGS. 24A and 24B and inFIGS. 24C and 24D, the tip of the pipette 52′ is press-sealed to theinjection side of the electrospray device 100. The electrospray device100 has a 10 μm diameter entrance orifice on the injection side, a 30 μminner diameter and a 60 μm outer diameter nozzle, a 15 μm nozzle wallthickness and a 150 μm nozzle depth. The recessed portion or the annulusextends 300 μm from the outer diameter of the nozzle. The voltageapplied to the fluid V_(fluid) introduced to the electrospray device andthus the nozzle voltage is 900 V. The voltage applied to the substrateV_(substrate) and thus the electrospray device is 0 V. The voltageapplied to the mass spectrometer which also serves as an extractingelectrode V_(extract) is approximately 40 V. The liquid sample waspumped using a syringe pump at a flow of 333 nL/min through the pipettetip pressed-sealed against the injection side of the electrospraydevice. The nozzle is approximately 5 mm from the ion-sampling orifice62 of the mass spectrometer 60. The ion-sampling orifice 62 of the massspectrometer 60 generally defines the acceptance region of the massspectrometer 60. The mass spectrometer for acquiring the data was theLCT Time-Of-Flight mass spectrometer of Micromass, Inc.

FIG. 24E shows a mass spectrum of 1 μg/mL PPG425 in 50% water, 50%methanol containing 0.1% formic acid, 0.1% acetonitrile and 2 mMammonium acetate. The data were collected at a flow rate of 333 nL/min.

Liquid Chromatography Device

In another aspect of the invention shown in the exploded perspective andcross-sectional views of FIGS. 25A and 25B, respectively, asilicon-based liquid chromatography device 400 generally comprises asilicon substrate or microchip 402 defining an introduction channel 404through the substrate 402 extending between an entrance orifice 406 on afirst surface 408 and a fluid reservoir 410, a separation channel 412extending between the reservoir 410 and an exit orifice 414, a pluralityof separation posts 416 along the separation channel 412, and a cover420 to provide an enclosure surface adjacent the cover 420 for thereservoir 410 and the separation channel 412 adjacent the cover 420.

The plurality of separation posts 416 extends from a side wall of theseparation channel 412 in a direction perpendicular to the fluid flowthough the separation channel 412. Preferably, one of the ends of eachseparation post 416 does not extend beyond and is preferably coplanar orlevel with the second surface 417. The separation channel 412 isfunctionally similar to the liquid chromatography column in thatcomponent separation occurs in the separation channel 412 where theplurality of separation posts 416 perform the liquid chromatographyfunction. Component separation occurs through the interaction of thefluid flowing through the separation channel 412 wherein the columnarseparation posts 416 provides the large surface area. The surfaces ofthe separation channel 412 and the separation posts 416 are preferablyprovided with an insulating layer to insulate the fluid in theseparation channel 412 from the substrate 402. Specifically, theseparation posts 416 are preferably oxidized silicon posts which may bechemically modified using known techniques in order to optimize theinteraction of the components of the sample fluid with the stationaryphase, the separation posts 416. In one embodiment, the separationchannel 412 extends beyond the separation posts 416 to the edge of thesubstrate 402 and terminating as the exit orifice 414.

The introduction channel, 404, the separation channel 412, the reservoir410 and the separation posts 416 may have any suitable cross-sectionalshapes such as circular and/or rectangular. Preferably, the separationposts 416 have the same cross-sectional shapes and sizes but maynonetheless have different cross-sectional shapes and/or sizes.

The liquid chromatography device 400 further comprises a layer ofsilicon dioxide 422 over the surfaces of the substrate of the cover 420and a layer of silicon dioxide 424 over the surfaces of the substrate402. The silicon dioxide layers 422, 424 electrically isolate a fluidcontained in the reservoir 410 and the separation channel 412 from thesubstrate 402 and the substrate of the cover 420. The silicon dioxidelayers 422, 424 are also relatively inactive and thus less likely tointeract with fluids in the reservoir 410 and the separation channel 412than bare silicon.

Depending on the specific application, the substrate 402 may provide asurface on which one or more conductive electrodes in electrical contactwith the fluid in the device 400 may be formed. For example, a reservoirelectrode 426 and/or an exit electrode 428 may be provided on the secondsurface 417 of the substrate 402 such that a corresponding electrodewould be in electrical contact with fluid in the reservoir 410 and nearthe exit orifice 414, respectively. A filling electrode 430 may also beprovided on the second surface 417 of the substrate 402 such that itwould be in electrical contact with fluid in the unpopulated portion 432of the separation channel 412 between the reservoir 410 and the firstoccurrence of separation posts 416. The shape, size and location alongthe fluidic flow path of each electrode on the substrate 402 may bedetermined by design considerations such as the distance betweenadjacent electrodes. Further, any or all of the electrodes may bealternatively or additionally formed on the bonding surface 425 of thecover 420. For example, the filling electrode 430 may be alternativelypositioned such that it would be in electrical contact with fluid in theseparation channel 412 adjacent the reservoir 410. Further, additionalelectrodes may be provided, for example, to create an arbitraryelectrical potential distribution along the fluidic flow path.

Providing two or more of the reservoir, filling and exit electrodesalong with electrical isolation of the fluid sample in the device 400from the substrate 402 and the substrate of the cover 420 allows for theapplication and sustenance of different (or same) electric potentials attwo or more different locations along the fluidic path. The differencein electric potentials at two or more different locations along thefluidic path causes fluidic motion to occur between the two or morelocations. Thus, these electrodes may facilitate the filling of thereservoir 410 and/or the driving of the fluid through the separationchannel 412.

Further, through appropriate layout design and fabrication processes,the substrate 402 and/or the cover 420 may also provide additionalfunctionalities such as pre-conditioning of the fluid prior to deliveryinto the reservoir 410, and/or conveying, analyzing, and/or otherwisetreating fluidic samples exiting from the separation channel 412. Thecover 420 may provide such additional functionality on either or bothsurfaces and/or the bulk of the cover 420.

The cover 420 may comprise a substrate 418 comprising silicon or anyother suitable material, such as glass plastics and/or polymers. Thespecific material for the cover 420 may depend upon, for example,whether direct observation of a fluoresced fluid is desired such thatglass may be more desirable and/or the consideration of the ease offabrication of the cover 420 by utilizing similar processing techniquesas for the substrate 402 such that silicon may be more desirable. Thecover .420 may be bonded or otherwise affixed to form a hermetic sealbetween the substrate 402 and the cover 420 in order to ensure theappropriate level of fluid containment and isolation. For example,several methods of bonding silicon to silicon or glass to silicon areknown in the art, including anodic bonding, sodium silicate bonding,eutectic bonding, and fusion bonding. The specific hermetic bondingmethod may depend on various factors such as the physical form of thesurfaces of the substrate 402 and the cover 420 and/or the applicationand functionality of the integrated system and/or the liquidchromatography device 400.

Dimensions of the liquid chromatography device 400 may be determinedaccording to various factors such as the specific application, thelayout design as well as the device with which it is to be interfaced orintegrated. The surface dimensions, i.e. the dimensions in the X and Ydirections, of the elements of the liquid chromatography device 400 maybe determined by layout design and through the corresponding photomasksused in fabrication. The depth or height, i.e. the dimension in the Zdirection, of the elements of the liquid chromatography device 400 maybe determined by the etch processes during fabrication, as describedbelow. The depth or height of the elements is independent of the surfacedimensions to a first-order approximation although the aspect ratiolimitations of the reactive-ion etch places constraints on the etchdepth, particularly with the small surface openings in the channel 412between the separation posts 416.

Further, the size, number, cross-sectional shape, spacing and placementof the separation posts 416 may also be determined by layout design toachieve the desired flow rate and to prevent low-resistance lines ofsight within the separation channel 412 to ensure adequate fluid-surfaceinteraction. Each separation post 416 may have the same or differentcharacteristics such as size and/or cross-sectional shape. Thecross-sectional shape of the posts may be chosen in layout design tooptimize fluid/boundary layer interactions at the post surfaces. Theseparation posts 416 may be placed in any desired pattern in theseparation channel 412, such as periodic, semi-periodic, or random.Close spacing of the separation posts 416 may be desirable formaximization of the surface interactions with the fluid. Similarly,minimizing the cross-sectional area of the separation posts 416 maypermit placement of greater number in the separation channel 412.However, the reduction of the cross-sectional area of the separationposts 416 is limited by the resulting reduction in the mechanicalstability necessary during processing.

Control of the size, number, cross-sectional shape, spacing andplacement of the separation posts 416 provides advantages overtraditional liquid chromatography as the traditional separation columnpacking materials have undesired dispersion in size distribution as wellas random spacing variations.

In one currently preferred embodiment, the substrate 402 of the liquidchromatography device 400 is approximately 250-600 μm in thickness, theseparation channel 412 has a depth of approximately 10 μm, therectangular reservoir 410 is approximately 1000 μm by 1000 μm resultingin a volume of approximately 10 nL. The depth of the reservoir 410 andthe separation channel 412 is limited by the height of the separationposts 416 which is in turn limited by the maximum etch aspect ratio. Thenearest-neighbor spacing of the separation posts 416 is preferably lessthan approximately 5 μm. The dimensions of the reservoir 410 determinethe volume of the fluid sample which can be used for the liquidchromatography separation and, as is evident, through the independentcontrol of surface dimensions and the depth, the reservoir 410 may bedesigned to have any desired volume. Preferably, the diameter of theentrance orifice 406 is 100 μm or less such that the fluid surfacetension would be sufficient to maintain the fluid in the reservoir 410to prevent leakage therefrom.

The silicon-based liquid chromatography device 400 reduces the size of atypical liquid chromatography device by nearly two orders of magnitude.The dimensional scaling may provide the advantage of significantlyreducing the mass of the analyte and/or the volume of the fluid samplerequired for accurate analysis. Further, by reducing a macroscopicseparation column and its packing materials to a monolithic device, theliquid chromatography device 400 can be a component of an on-chipintegrated system.

Further, all features such as the reservoir, the separation channel andthe separation posts are recessed from the substrate 402. The portion ofthe substrate 402 exterior to the reservoir and the separation channelthus serves to physically protect the separation posts from casualabrasion and stress fracture in handling and subsequent bonding of thesubstrate 402 and the cover 420. Because the posts are integral with thesubstrate, the posts are inherently stable and thus allow for the use ofa pressurized system without the risk of damage to the stationary phasewhich may otherwise result with the use of conventional packingmaterials in conventional high-performance liquid chromatographysystems.

An upstream fluid delivery system, such as a micropipette, piezoelectricpipette or small capillary, may be press-sealed onto the exteriorsurface of the liquid chromatography device 400 such that the pipette orcapillary is concentric with the entrance orifice 406. Optionally, theliquid chromatography device may provide a collar (not shown) tofacilitate the mating and affixing of the fluid delivery device to theliquid chromatography device similar to the mating collar of theelectrospray device as discussed with reference to FIG. 21B.

To operate the liquid chromatography device 400, the fluid reservoir 410may first be filled with a sample fluid by injecting the fluid from afluid delivery device through the introduction channel 404 via theentrance orifice 406. Any suitable fluid delivery device such as amicropipette, a piezoelectric pipette or a small capillary may beutilized. The volume of the sample fluid injected into the liquidchromatography device 400 may be up to approximately the volume of thereservoir 410 plus a relatively small volume remaining in theintroduction channel 404.

The filling of the reservoir 410 may be facilitated by applying anappropriate potential voltage difference between the reservoir electrode426 and the filling electrode 430, such as approximately 1000 V/cm ofintroduction channel 404. In particular, a volume of the fluid is firstintroduced into the reservoir 410 through the introduction channel 404via the entrance orifice 406 to coat or prime the surfaces of thereservoir 410 and the introduction channel 404 by capillary action toallow for electrical contact between the fluid and the reservoir andfilling electrodes 426, 430. Where the filling electrode 430 ispositioned in a portion of the separation channel 412 unpopulated byseparation posts 416, the filling electrode 430 also facilitates thefilling of the portion of the channel 412 between the reservoir 410 andthe filling electrode 430.

After filling the reservoir 410 with an appropriate volume of the samplefluid, any suitable method may then be utilized to drive the fluid fromthe reservoir 410 into the separation channel 412. For example, thefluid may be driven from the filled reservoir 410 through the separationchannel 412 by applying hydrostatic pressure to the reservoir 410 viathe entrance orifice 406.

Alternatively or additionally, the fluid may be driven through theseparation channel 412 by applying a suitable electrokinetic potentialvoltage difference between the reservoir electrode 426 and the exitelectrode 428 to generate electrophoretic or electroosmotic fluidicmotion. Preferably, the electric potential difference is approximately1000 V/cm of separation channel length. Of course, any other suitablemethods of inducing fluidic motion may be utilized. Pressure-driven andvoltage-driven flow effect different separation efficiencies. Thus,depending upon the application, one or both may be utilized.

Fluid then exits from the separation channel 412 through the exitorifice 414 to, for example, a capillary 434, which has an off-chipinterconnection with the exit orifice 414, as shown in FIG. 26.Alternatively, as shown in FIG. 27, the liquid chromatography device 400may perform separation on the fluid from reservoir 410 such thatselected analytes from the separation performed by posts 416 passesthrough unpopulated channel 436 to another on-chip device 438, such asfor analysis and/or mixing, while the remainder of the fluid is directedto the waste reservoir 439. The unpopulated channel 436 may be a merecontinuation of the separation channel 412 of the liquid chromatographydevice 400 or a channel separate from the separation channel 412.

Two or more fluid samples may be driven through the liquidchromatography device 400 by successively filling the reservoir anddriving the fluid through the separation channel 412. For example, incertain applications, it may be desirable or necessary to first coat thesurfaces of the separation posts 416 with one or more reagents and thenpass an analyte sample over the conditioned separation posts 416.

Various modifications may be made to the liquid chromatography devicedescribe above. For example, as shown in FIG. 28, rather than definingthe entrance orifice and the introduction channel in the substrate, theliquid chromatography device 400′ may provide an introduction channel404′ in the cover 420′ such that the entrance orifice 406′ is defined onan exterior surface of the cover 420′. Further, the cover 420′ maydefine an exit channel 413 between an exit orifice 414′ defined on anexterior surface of the cover 420′ and a separation channel 412′ whichterminates within the substrate 402′.

In another variation, an additional introduction channel 440 andentrance orifice 442 may be defined in the substrate 402″, as shown inFIG. 29, or in the cover (not shown). The additional introductionchannel 440 introduces fluid to the separation channel 412″ such thatthe fluid from the additional introduction channel 440 intersects thepath of fluid flow from the reservoir 410 through the unpopulatedportion 432″ of the separation channel 412″. The fluid reservoir 410 maybe utilized as a buffer for an eluent and the additional introductionchannel 440 may be utilized to introduce the fluid sample to theseparation channel 412″. Further, the additional entrance orifice 442may be utilized to introduce several fluid samples in succession intothe separation channel 412″. For example, in certain applications, itmay be necessary to first coat the surfaces of the separation posts 416with one reagent and then pass an analyte over the conditioned: surfacesof the separation posts 416.

Referring now to FIGS. 30-35, although the liquid chromatography devicehas been described as comprising a single reservoir and a singleseparation channel, the monolithic liquid chromatography device may beeasily adapted and modified to comprise multiples of the liquidchromatography device and/or multiple entrance orifices, exit orifices,reservoirs and/or separation channels. In each of the variations, any orall of the reservoir(s), separation channel(s), and separation posts mayhave different dimensions and/or shapes.

For example, multiple reservoir-separation channel combinations may beprovided on a single chip. In particular, as shown in FIG. 30, areservoir 410A may feed into a separation channel 412A having separationposts 416A and another reservoir 410B may feed into another separationchannel 412B having separation posts 416B.

In another variation as shown in FIG. 31, a single reservoir 410C mayfeed multiple separation channels 412C, 412D. Each of separationchannels 412C, 412D may have therein separation posts 416C, 416D,respectively, which may have the same or different properties, such asnumber, size and shape. Another channel 412E may be provided as a nullchannel be utilized as a basis of comparison to the output from theseparation channel(s) populated by separation posts. Alternatively, allof the channels 412C, 412D, 412E may be separation channels havingseparation posts.

Referring now to FIG. 32, fluid from multiple reservoirs 410E and 410Fmay feed into a single separation channel 412F via connecting channels444E, 444F, respectively. The connecting channels 444E, 444F arepreferably unpopulated by separation posts to facilitate the mixing ofthe fluid samples from the reservoirs 410E, 410F prior to passagethrough the separation channel 412F. The mixing of samples may beutilized to condition the primary sample of interest prior to separationor to effect a reaction between the samples prior to passage through thepopulated portion of the separation channel 412F. Alternatively, fluidsuch as a conditioning fluid from one reservoir 410E may flow throughthe separation channel 412F in order to condition the surfaces of theseparation posts 416F prior to the passage of the other sample such asan analyte sample from the other reservoir 410F. Although the separationposts 416F are shown as having different cross-sections, separationposts 416F may have the same size and cross-sectional shape.

Alternatively, in addition to having fluid from multiple reservoirs feedinto a single separation channel via connecting channels, fluid fromanother reservoir may be introduced to the fluid flow along theseparation channel, before and/or after the fluid has passed through thepopulated portion of the separation channel. For example, FIG. 33 showsthat the fluid from multiple reservoirs 410G, 410H may be fed into asingle separation channel 412G via connecting channels 444G, 444H,respectively, and fluid from another reservoir 410I may be introduced tothe fluid flow along the separation channel 412G after the fluid haspassed the separation posts 416G. FIG. 34 shows that the fluid frommultiple reservoirs 410J, 410K may be fed into a single separationchannel 412J via connecting channels 444J, 444K, respectively, and fluidfrom another reservoir 410L may be introduced to the fluid flow alongthe separation channel 412J prior to the fluid passing the separationposts 416J.

For devices having multiple reservoirs and/or multiple channels,separate electrodes may be provided for each reservoir and/or for eachchannel, for example, in the unpopulated portion of the channel upstreamfrom the separation posts and/or near the exit of the channel. Suchprovision of separate electrodes allow for the separate and independentcontrol of the fluidic flow for filling each reservoir and/or fordriving the fluid through the separation channel.

The electric control may be simplified by having one common reservoirelectrode, one common filling electrode, and/or one exit electrode amongthe multiple reservoirs and/or multiple channels. For example, each ofthe multiple reservoirs may be separately filled by applying a firstvoltage to the common reservoir electrode and a second voltage,different from the first voltage, to the filling electrode correspondingto the reservoir to be filled while applying the first voltage to eachof the other filling electrodes. As is evident, the multiple reservoirsmay be simultaneously filled by applying a first voltage to the commonreservoir electrode and a second, different voltage to each of thefilling electrodes. Similarly, fluid may be separately driven througheach of the multiple channels by applying a third voltage to the commonreservoir electrode while applying a fourth voltage, different from thethird voltage, to the exit electrode corresponding to the channelthrough which fluid is to be driven and the third voltage to each of theother exit electrodes.

In yet another variation shown in FIG. 35, in addition to a samplereservoir 410M and separation posts 416M, a plurality of posts 416L maybe provided in a channel 412M upstream from the separation posts 416Mfor providing additional functionality such as solid-phase extraction(SPE) for sample pretreatment. The SPE posts 416L may be the same,similar to or different from the separation posts 416M simply by varyingthe layout design. The SPE posts 416L may provide surface functionalitydifferent from that of the separation posts 416M. Alternatively, ratherthan providing a sample reservoir, an introduction channel (not shown)may be utilized to introduce a fluidic sample directly in the channel412M by allowing direct injection of the sample therein. Further,reservoirs 410N, 410P may be provided to contain fluidic buffersnecessary for sample pretreatment upstream of the posts 416L. Forexample, an eluent reservoir may be provided for eluting analytes and awash reservoir may be provided for sample cleanup.

After the fluid samples pass the SPE posts 416L, waste products from,for example, the solid-phase extraction process may be directed into awaste reservoir 410Q. In particular, during the SPE process, voltagedifferences may be applied between or amongst reservoirs 410M, 410N,410P, and 410Q such that a portion of the fluid from reservoirs 410M,410N is directed to waste reservoir 410Q while the remaining portion ofthe fluid from reservoir 410M remain on the SPE posts 416L. Material maythen be washed off of the SPE posts 416L by directing fluid from, forexample, reservoir 410P through channel 412M for separation of theextracted material by separation posts 416M. Additional reservoirs 410R,410S downstream of the waste reservoir 410Q and upstream of theseparation posts 416M may be provided to contain gradient elution ofanalytes in one reservoir and a diluent in the other reservoir. Gradientelution facilitates chromatography by changing the mobile phasecomposition, i.e. the polarity to facilitate analyte interactions withthe stationary phase, and thus facilitate separation of the analytes. Inaddition, the diluent provides the correct polarity of the solution forthe next separation.

Liquid Chromatography Device Fabrication Procedure

The fabrication of the liquid chromatography device of the presentinvention will now be explained with reference to FIGS. 36A-46B. Theliquid chromatography device is preferably fabricated as a monolithicsilicon micro device utilizing established, well-controlled thin-filmsilicon processing techniques such as thermal oxidation,photolithography, reactive-ion etching (RIE), ion implantation, andmetal deposition. Fabrication using such silicon processing techniquesfacilitates massively parallel processing of similar devices, is time-and cost-efficient, allows for tighter control of critical dimensions,is easily reproducible, and results in a wholly integral device, therebyeliminating any assembly requirements. Manipulation of separatecomponents and/or subassemblies to build an liquid chromatography devicewith high reliability and yield is not desirable and may not be possibleat the micrometer dimensions required for efficient separation.

Further, the fabrication sequence may be easily extended to createphysical aspects or features to facilitate interfacing, integrationand/or connection with devices having other functionalities or tofacilitate integration with a fluid delivery subsystem to create asingle integrated system. Consequently, the liquid chromatography devicemay be fabricated and utilized as a disposable device, therebyeliminating the need for column regeneration and eliminating the risksof sample cross-contamination.

Referring to the plan and cross-sectional views, respectively, of FIGS.36A and 36B, a silicon wafer separation substrate 500, double-sidepolished and approximately 250-600 μm in thickness, is subjected to anelevated temperature in an oxidizing ambient to grow a layer or film ofsilicon dioxide 502 on the reservoir side 503 and a layer or film ofsilicon dioxide 504 on the back side 505 of the separation substrate500. Each of the resulting silicon dioxide layers 502, 504 has athickness of approximately 1-2 μm. The silicon dioxide layers 502, 504provide electrical isolation and also serve as masks for subsequentselective etching of certain areas of the separation substrate 500.

A film of positive-working photoresist 506 is deposited on the silicondioxide layer 502 on the reservoir side 503 of the separation substrate500. Certain areas of the photoresist 506 corresponding to thereservoir, separation channel and separation posts which will besubsequently etched are selectively exposed through a mask by an opticallithographic exposure tool passing short-wavelength light, such as blueor near-ultraviolet at wavelengths of 365, 405, or 436 nanometers.

Referring to the plan and cross-sectional views, respectively, of FIGS.37A and 37B, after development of the photoresist 506, the exposed areas508, 509, 510 of the photoresist corresponding to the reservoir,separation posts and channel, respectively, are removed and open to theunderlying silicon dioxide layer 502 while the unexposed areas remainprotected by photoresist 506′. The exposed areas 508, 509, 510 of thesilicon dioxide layer 502 are then etched by a fluorine-based plasmawith a high degree of anisotropy and selectivity to the protectivephotoresist 506′ until the silicon separation substrate 500 is reached.The remaining photoresist is removed in an oxygen plasma or in anactively oxidizing chemical bath like sulfuric acid (H₂SO₄) activatedwith hydrogen peroxide (H₂O₂).

As shown in the cross-sectional view of FIG. 38, the reservoir 410, theseparation channel 412, and the separation posts 416 in the separationchannel 412 are vertically formed in the silicon separation substrate500 by another fluorine-based etch. Preferably, the reservoir 410 andthe separation channel 412 have the same depth controlled by the etchtime at a known etch rate. The simultaneous formation of the reservoir410 and the channel 412 ensures uniform depth such that there are nodiscontinuities in the fluid-constraining surfaces to impede the fluidflow. The depth of the reservoir 410 and the channel 412 is preferablybetween approximately 5-20 μm and more preferably approximately 10 μm.The etch can reliably and reproducibly be executed to produce an aspectratio (etch depth to width) of up to 30:1. Although not shown, any otherreservoirs and/or channels, populated or unpopulated, may also be formedby this etch sequence.

A film of positive-working photoresist is then deposited over thesilicon dioxide layer 502 and the exposed separation substrate 500 onthe reservoir side 503 of the separation substrate 500. An area of thephotoresist corresponding to the introduction channel which will besubsequently etched is selectively exposed through a mask by an opticallithographic exposure tool passing short-wavelength light, such as blueor near-ultraviolet at wavelengths of 365, 405, or 436 nanometers. Afterdevelopment of the photoresist, the exposed area of the photoresistcorresponding to the introduction channel is removed and open to theunderlying separation substrate 500 while the unexposed areas remainprotected by the photoresist.

As shown in the plan and cross-sectional views of FIGS. 39A and 39B,respectively, the exposed area of the separation substrate 500 is thenvertically etched by a fluorine-based plasma with a high degree ofanisotropy and selectivity to the protective photoresist until thesilicon dioxide layer 504 on back side 505 is reached. Thus, a portionof the introduction channel 404 is formed through the separationsubstrate 500. The remaining photoresist is removed in an oxygen plasmaor in an actively oxidizing chemical bath like sulfuric acid (H₂SO₄)activated with hydrogen peroxide (H₂O₂). The silicon dioxide layer 504on the back side 505 may then be removed by, for example, an unpatternedetch in a fluorine-based plasma.

Alternatively, as shown in FIGS. 40A and 40B, the introduction channel404 may be formed by etching from both the reservoir side 503 and theback side 505 of the substrate 500. After performing a vertical etchthough a portion of the substrate 500 to form a portion of theintroduction channel 404 in a manner similar to that described above, afilm of positive-working photoresist 512 is deposited on the silicondioxide layer 504 on the back side 505 of the separation substrate 500.Patterns on the back side 505 may be aligned to those previously formedon the reservoir side 503 of the separation substrate 500. Becausesilicon and its oxide are inherently relatively transparent toB light inthe infrared wavelength range of the spectrum, i.e. approximately700-1000 nanometers, the extant pattern on the reservoir side 503 can bedistinguished with sufficient clarity by illuminating the separationsubstrate 500 from the patterned reservoir side 503 with infrared light.Thus, the mask for the back side 505 can be aligned within requiredtolerances. Upon alignment, an area of the photoresist 512 correspondingto the entrance orifice and the introduction channel which will besubsequently etched is selectively exposed through a mask by an opticallithographic exposure tool passing short-wavelength light, such as blueor near-ultraviolet at wavelengths of 365, 405, or 436 nanometers.

After development of the photoresist 512, the exposed area 514 of thephotoresist corresponding to the entrance orifice is removed to exposethe underlying silicon dioxide layer 504 on the back side 505 of theseparation substrate 500 while the unexposed areas remain protected bythe photoresist 512. The exposed area 514 of the silicon dioxide layer504 is then etched by a fluorine-based plasma with a high degree ofanisotropy and selectivity to the protective photoresist 512 until thesubstrate 500 is reached. The remaining photoresist provides additionalmasking during a subsequent fluorine-based silicon etch to verticallyetch the backside portion of the introduction channel. Thus, athrough-substrate introduction channel 404 is complete. The remainingphotoresist is removed in an oxygen plasma or in an actively oxidizingchemical bath like sulfuric acid (H₂SO₄) activated with hydrogenperoxide (H₂O₂).

Preferably, the introduction channel 404 has the same diameter as theentrance orifice. A practical limit on etch aspect ratio of 30:1constrains the diameter of the entrance orifice being etched to beapproximately 10 μm or greater for substrates of approximately 300 μmthickness. Preferably, the entrance orifice 406 and the introductionchannel 404 are approximately 100 μm in diameter due to practicalconsiderations. For example, the etch aspect ratio imposes a minimumdiameter, and the diameter is preferably sufficiently large to enableease of filling the reservoir 410 yet sufficiently small to ensure afluid surface tension to prevent the fluid from leaking out of thereservoir 410.

Alternatively, both the introduction channel and the entrance orificemay be formed by etching from the back side 505 of the separationsubstrate 500. This may be preferable as it may be difficult tosatisfactorily coat the separation posts 416 with photoresist. Further,this may be desirable depending on the application of the device, e.g.the external sample delivery system, the desired chip handling devices,the interfacing with other devices, chip-based or non-chip based, and/orthe packaging considerations of the chip. Referring to thecross-sectional view of FIG. 41, after the reservoir, separation channeland the separation posts are etched in the separation substrate 500(shown in FIG. 38), a film of positive-working photoresist 516 isdeposited on the silicon dioxide layer 504 on the back side 505 of theseparation substrate 500. Patterns on the back side 505 may be alignedto those previously formed on the reservoir side 503 of the separationsubstrate 500 by illuminating the separation substrate 500 from thepatterned reservoir side 503 with infrared light, as described above.Upon alignment, an area of the photoresist 516 corresponding to theentrance orifice which will be subsequently etched is selectivelyexposed through a mask by an optical lithographic exposure tool passingshort-wavelength light, such as blue or near-ultraviolet at wavelengthsof 365, 405, or 436 nanometers.

After development of the photoresist 516, the exposed area 518 of thephotoresist 516 corresponding to the entrance orifice is removed toexpose the underlying silicon dioxide layer 504 on the back side 505 ofthe separation substrate 500. The exposed area 518 of the silicondioxide layer 504 is then etched by a fluorine-based plasma with a highdegree of anisotropy and selectivity to the protective photoresist 512until the silicon separation substrate 500 is reached. The remainingphotoresist is left in place to provide additional masking during thesubsequent etch through the silicon separation substrate 500.

Referring now to the cross-sectional view of FIG. 42, the introductionchannel 404 is vertically formed through the silicon separationsubstrate 500 by another fluorine-based etch. The introduction channel404 is completed by etching through the separation substrate 500 untilthe reservoir 410 is reached. Thus, the introduction channel 404 extendsthrough the separation substrate 500 between the entrance orifice 406 onthe back side 505 of the separation substrate 500 and the reservoir 410.The remaining photoresist is removed in an oxygen plasma or in anactively oxidizing chemical bath like sulfuric acid (H₂SO₄) activatedwith hydrogen peroxide (H₂O₂).

Oxidation for Surface Passivation and Fluid Isolation

As shown in the cross-sectional view of FIG. 43, a layer of silicondioxide 522 is grown on all silicon surfaces of the substrate 500 bysubjecting the silicon substrate 500 to elevated temperature in anoxidizing ambient. For example, the oxidizing ambient may be anultra-pure steam produced by oxidation of hydrogen for a silicon dioxidethickness greater than approximately several hundred nanometers or pureoxygen for a silicon dioxide thickness of approximately several hundrednanometers or less. The layer of silicon dioxide 522 over all siliconsurfaces of the separation substrate 500 electrically isolates a fluidin the channel from the silicon substrate 500 and permits theapplication and sustenance of an electric potential difference betweenthe reservoir and the exit of the separation channel, between thereservoir and an unpopulated portion of the separation channel near thereservoir to facilitate in filling the reservoir and/or between otherpoints along the fluid flow path. Thus, the application and sustenanceof a significant voltage across the fluid sample may be achieved.Further, oxidation renders a surface inactive relative to a bare siliconsurface, resulting in surface passivation.

All silicon surfaces are oxidized to form silicon dioxide with athickness that is controllable through choice of temperature and time ofoxidation. The final thickness of the silicon dioxide can be selected toprovide the desired degree of electrical isolation in the device, wherea thicker layer of silicon dioxide provides a greater resistance toelectrical breakdown.

Photolithography and reactive-ion etching limit the layout design ofseparation post diameters and inter-post spacing to greater thanapproximately 1 μm. However, because the thermal oxidation processconsumes approximately 0.44 μm of silicon to form each micrometer ofsilicon dioxide, the thermal oxidation process results in a volumetricexpansion. This volumetric expansion may be utilized to reduce thespacing between the separation posts 416 to sub-micrometer dimensions.For example, with a layout inter-post spacing of approximately 1.5 μm,oxidation producing a 1 μm silicon dioxide film or layer would result ina nearest-neighbor spacing of approximately 0.5 μm. Further, because theoxidation process is well-controlled, separation post dimensions,including the inter-post spacing, in the sub-micrometer regime can beformed reproducibly and in a high yielding manner.

FIGS. 44A, 44B and 44C show scanning electron microscope photographs anddesign layout of portions of fabricated liquid chromatography devices.FIG. 44A shows a design layout of a portion of a reservoir andseparation posts in a portion of a separation channel where theseparation posts have rectangular cross-sectional shape. FIG. 44B showsseparation posts in a portion of a separation channel, the separationposts having a circular cross-sectional shape and a diameter andinter-post spacing of approximately 1 μm. FIG. 44C shows separationposts in a portion of a separation channel, the separation posts havinga rectangular or square cross-sectional shape with a dimension of 2 μmand inter-post spacing of approximately 1 μm.

In a variation, the entrance orifice and the introduction channel forfilling the fluid reservoir may be formed in the cover substrate 524after a layer of silicon dioxide 525 is grown on all surfaces of thecover substrate 524, rather than in the substrate 500. As shown in FIG.45, the cover substrate 524 may be bonded to the reservoir side 503 ofthe separation substrate 500. The entrance orifice 406′ and theintroduction channel 404′ may be formed in the cover substrate 524 afteralignment with respect to the reservoir 410. The entrance orifice 406′and the introduction channel 404′ may be formed in the same or similarmanner as described above by utilizing lithography to define theentrance orifice pattern and reactive-ion etching to create the entranceorifice and the through-cover introduction channel. The cover substrate524 is again subjected to elevated temperature in an oxidizing ambientto grow a layer of oxide on the surface of the introduction channel404′. Further, the introduction channel 404′ may be formed from one ortwo sides of the cover substrate 524. If channel 404′ is formed from twosides of the cover substrate, the cover substrate 524 may be bonded tosubstrate 500 after forming the channel 404′ and after oxidation of thechannel surface. One advantage of defining the entrance orifice on thesame side of the completed liquid chromatography device as the reservoirand separation channel is that the back side of the substrate 500 isthen free from any features and may then be bonded to a protectivepackage without special provision for filling the reservoir through anentrance orifice defined on the back-side of the substrate.

Metallization for Fluid Flow Control

FIGS. 46A and 46B illustrate the formation of a reservoir, a filling,and an exit electrode as well as conductive lines or wires connectingthe electrodes to bond pads in the cover substrate 526, preferablycomprising glass and/or silicon. The cover substrate 526 shown in FIGS.46A and 46B does not provide an entrance orifice or an introductionchannel although the metallization process described herein may beeasily adapted for a cover substrate providing an entrance orifice andan introduction channel.

As shown in the plan and cross-sectional view of FIGS. 46A and 46B,respectively, prior to the depositing of conductive material on thecover substrate 526, all surfaces of the cover substrate 526 aresubjected to thermal oxidization in a manner that is the same as orsimilar to the process described above to create a film or layer ofsilicon dioxide 528. Such oxidization is not performed where the coversubstrate 526 comprises glass.

The silicon dioxide layer 528 provides a surface on which conductiveelectrodes may be formed. The thickness of the silicon dioxide layer 528is controllable through the oxidation temperature and time and the finalthickness can be selected to provide the desired degree of electricalisolation, where a thicker layer of silicon dioxide provides a greaterresistance to electrical breakdown. The silicon dioxide layer 528electrically isolates all electrodes from the cover substrate 526 andisolates the fluid in the reservoir and the channel of the liquidchromatography device from the cover substrate 526. The ability toisolate the fluid from the cover substrate 526 complements theelectrical isolation provided in the separation substrate throughoxidation and ensures the complete electrical isolation of the fluidfrom both the separation substrate and the cover substrate 526. Thecomplete electrical isolation of the sample fluid from both substratesallows for the application of electric potential differences betweenspatially separated locations in the fluidic flow path resulting incontrol of the fluid flow through the path.

The cover substrate 528 may be cleaned after oxidation utilizing anoxidizing solution such as an actively oxidizing chemical bath, forexample, sulfuric acid (H₂SO₄) activated with hydrogen peroxide (H₂O₂).The cover substrate 528 is then thoroughly rinsed to eliminate organiccontaminants and particulates. A layer of conductive material 530 suchas aluminum is then deposited by any suitable method such as by DCmagnetron sputtering in an argon ambient. The thickness of the aluminumis preferably approximately 3000 Å, although shown having a largerthickness for clarity. Although aluminum is utilized in the fabricationsequence described herein, any type of highly conductive material suchas other metals, metallic multi-layers, silicides, conductive polymers,and conductive ceramics like indium tin oxide (ITO) may be utilized forthe electrodes. The surface preparation for satisfactory adhesion mayvary depending on the specific electrode material used. For adhere asaluminum does not generally adhere well to native silicon.

A film of positive-working photoresist 532 is then deposited over thesurface of the conductive material 530. Areas of the photoresist layer532 corresponding to areas surrounding the electrodes (shown) andconductive lines or wires and bond pads which will be subsequentlyetched are selectively exposed through a mask by an optical lithographicexposure tool passing short-wavelength light, such as blue ornear-ultraviolet at wavelengths of 365, 405, or 436 nanometers.

After development of the photoresist 532, the exposed areas of thephotoresist are removed, leaving opening to the underlying aluminumconductive layer 530 while the unexposed areas 534, 536, 538corresponding to the reservoir, filling and exit electrodes,respectively, as well as conductive lines or wires and bond pads remainprotected by the photoresist. The conductive electrodes and thelines/bond pads may be etched, such as by a wet chemical etch or areactive-ion etch, as appropriate for the particular conductivematerial. The etch is selective to the underlying silicon dioxide layer528 or is terminated upon reaching the silicon dioxide layer 528 asdetermined by the etch time and rate. The remaining photoresist isremoved in an oxygen plasma or in a solvent bath such as acetone. Thefabrication sequence thus results in physically and electricallyseparate islands of conductive electrodes, lines and bond pads accordingto the pattern designed in the mask.

The cover substrate may be larger than the separation substrate to allowaccess to the bond pads and/or directly to the electrodes for theapplication of potential voltage(s) to the electrode(s). As shown inFIG. 46C, the cover substrate 526′ is larger than the separationsubstrate such that the separation substrate only extends to dashed line540 relative to the cover substrate 526′. Conductive lead-throughs suchas connecting metal lines 542, 544 and 546 extend from the reservoir,filling and exit electrodes, 534, 536, 538, respectively, and enable theapplication of potential voltage(s) to the electrode(s).

Alternatively, a metal lead may be formed from each electrode to anotherwise unpatterned area of the separation substrate such that athrough-substrate access channel formed in the cover substrate andfilled with a conductive material by chemical vapor deposition (CVD)allows access to the electrode(s). As an alternative to chemical vapordeposition, the sidewalls of the through-substrate access channel may besloped, for example by KOH etch, to facilitate continuous deposition ofa conductive material thereon, thereby providing an electricallycontinuous path from the separation substrate to the top of the coversubstrate where potential voltages can be applied. In these variations,the separation and the cover substrates may be of the same size.

Although the electrodes are preferably provided on a surface of thecover substrate, the electrodes may be alternatively and/or additionallyprovided on the separation substrate by appropriate modifications to theabove-described fabrication process. For example, in such a variation,the side walls of the reservoir are preferably not at a 90° anglerelative to the bottom wall and can be formed at least in part by, forexample, a wet chemical potassium hydroxide (KOH) etch. The slopedreservoir side walls allow for the deposition of a conductive materialthereon. In another variation, the electrodes may also be formed by adamascene process, known in the art of semiconductor fabrication. Thedamascene process provides the advantage of a planar surface without thestep up and step down surface topography presented by a bond line or padand thus facilitates the bonding of the separation and cover substrate,as described below.

The above described fabrication sequence for the liquid chromatographydevice may be easily adapted to and is applicable for the simultaneousfabrication of a monolithic system comprising multiple liquidchromatography devices including multiple reservoirs and/or multipleseparation channels as described above embodied in a single monolithicsubstrate.

Further, although the fabrication sequence is described in terms offabricating a single liquid chromatography device, the fabricationsequence facilitates and allows for massively parallel processing ofsimilar devices. The multiple liquid chromatography devices or systemsfabricated by massively parallel processing on a single wafer may thenbe cut or otherwise separated into multiple devices or systems.

Although control of the liquid chromatography device has been describedabove as comprising reservoir, filling and exit electrodes, any suitablecombination of such and/or other electrodes in electrical contact withthe fluid in the fluid path may be provided and easily fabricated bymodifying the layout design. Further, any or all of the electrodes maybe additionally or alternatively provided in the separation substrate.Electrodes may be formed in the separation substrate by modifying thefabrication sequence to include additional steps similar to or the sameas the steps as described above with respect to the formation of theelectrodes in the cover substrate.

Bonding Cover Substrate to Separation Substrate

As described above, the cover substrate is preferably hermeticallybonded by any suitable method to the separation substrate forcontainment and isolation of the fluid in the liquid chromatographydevice. Examples of bonding silicon to silicon or glass to siliconinclude anodic bonding, sodium silicate bonding, eutectic bonding, andfusion bonding.

For example, to bond the separation substrate to a glass cover substrateby anodic bonding, the separation substrate and cover substrate areheated to approximately 400° C. and a voltage of 400-1200 Volts isapplied, with the separation substrate chosen as the anode (the higherpotential). Further, as the required bonding voltage depends on thesurface oxide thickness, it may be desirable to remove the oxide film orlayer from the back side 505 of the separation substrate prior to thebonding process in order to reduce the required bonding voltage. Theoxide film or layer maybe removed by, for example, an unpatterned etchin a fluorine-based plasma. The etch is continued until the entire oxidelayer has been removed, and the degree of over-etch is unimportant.Thus, the etch is easily controlled and high-yielding.

Critical considerations in any of the bonding methods include thealignment of features in the separation and the cover substrates toensure proper functioning of the liquid chromatography device afterbonding and the provision in layout design for conductive lead-throughssuch as the bond pads and/or metal lines so that the electrodes (if any)are accessible from outside the liquid chromatography device. Anothercritical consideration is the topography created through the fabricationsequence which may compromise the ability of the bonding method tohermetically seal the separation and cover substrates. For example, thestep up and step down in the surface topography presented by a metalline or pad may be particularly difficult to form a seal therearound asthe silicon or glass does not readily deform to conform to the shape ofthe metal line or pad, leaving a void near the interface between themetal and the oxide.

Integration of Liquid Chromatography and Electrospray Devices on a Chip

The cross-sectional schematic view of FIG. 47 shows a liquidchromatography-electrospray system 600 comprising a liquidchromatography device 602 of the present invention integrated with anelectrospray device 620 of the present invention such that a homogeneousinterface is formed between the exit orifice 614 of the liquidchromatography device 602 and the entrance orifice 622 of theelectrospray device 620. The single integrated system 600 allows for thefluid exiting the exit orifice 614 of the liquid chromatography device602 to be delivered on-chip to the entrance orifice 622 of theelectrospray device 620 in order to generate an electrospray.

As shown in FIG. 47, the entrance orifice 606 and the introductionchannel 604 of the liquid chromatography device, 602 are formed in thecover substrate 608 along with the electrospray device 620.Alternatively, the liquid chromatography entrance orifice and theintroduction channel may be formed in the separation substrate.

Fluid at the electrospray nozzle entrance 622 is at the exit voltageapplied to the exit electrode 610 in the separation channel 612 near theliquid chromatography exit orifice 614. Thus, an electrospray entranceelectrode is not necessary.

The single integrated system 600 provides the advantage of minimizing oreliminating extra fluid volume to reduce the risk of undesired fluidchanges, such as by reactions and/or mixing. The single integratedsystem 600 also provides the advantage of eliminating the need forunreliable handling and attachment of components at the microscopiclevel and of minimizing or eliminating fluid leakage by containing thefluid within one integrated system.

The integrated liquid chromatography-electrospray system 600 may beutilized to deliver liquid samples to the sampling orifice of a massspectrometer. The sampling orifice of the mass spectrometer may serve asan extraction electrode in the electrospray process when held at anappropriate voltage relative to the voltage of the electrospray nozzle624. The liquid chromatography-electrospray system 600 may be positionedwithin 10 mm of the sampling orifice of the mass spectrometer forefficient extraction of the fluid from the electrospray nozzle 624.

Multiple Liquid Chromatography-electrospray Systems on a Single Chip

Multiples of the liquid chromatography-electrospray system 600 may beformed on a single chip to deliver a multiplicity of samples to a commonpoint for subsequent sequential analysis. For example, FIG. 48 shows aplan view of multiple liquid chromatography-electrospray systems 600 ona single chip 650 and FIG. 49 shows a detailed view of area A of systems600 with the separation channels shown in phantom and without therecessed portions for purposes of clarity. As shown, the multiplenozzles 624 of the electrospray devices 620 may be radially positionedabout a circle having a relatively small diameter near the center of thesingle chip 650. The dimensions of the electrospray nozzles and theliquid chromatography channels limit the radius at which multiplenozzles are positioned on the multi-system chip 650. For example, themulti-system chip may provide 96 nozzles with widths of up to 50 μmpositioned around a circle 2 mm in diameter such that the spacingbetween each pair of nozzles is approximately 65 μm.

Alternatively, an array of multiple electrospray devices without liquidchromatography devices may be formed on a single chip to deliver amultiplicity of samples to a common point for subsequent sequentialanalysis. The nozzles may be similarly radially positioned about acircle having a relatively small diameter near the center of the chip.The array of electrospray devices on a single microchip may beintegrated upstream with multiple fluid delivery devices such asseparation devices fabricated on a single microchip. For example, anarray of radially distributed exit orifices of a radially distributedarray of micro liquid chromatography columns may be integrated withradially distributed entrance orifices of electrospray devices such thatthe nozzles are arranged at a small radius near the orifice of a massspectrometer. Thus, the electrospray devices may be utilized for rapidsequential analysis of multiple sample fluids. However, depending uponthe specific application and/or the capabilities of the downstream massspectrometer (or other downstream device), the multiples of theelectrospray devices may be utilized one at a time or simultaneously,either all or a portion of the electrospray devices, to generate one ormore electrosprays. In other words, the multiples of the electrospraydevices may be operated in parallel, staggered or individually.

The single multi-system chip 650 may be fabricated entirely in siliconsubstrates, thereby taking advantage of well-developed siliconprocessing techniques described above. Such processing techniques allowthe single multi-system chip 650 to be fabricated in a cost-effectivemanner, resulting in a cost performance that is consistent with use as adisposable device to eliminate cross-sample contamination. Furthermore,because the dimensions and positions of the liquidchromatography-electrospray systems are determined through layout designrather than through processing, the layout design may be easily adaptedto fabricate multiple liquid chromatography-electrospray systems on asingle chip.

Interface of a Multi-system Chip to mass Spectrometer

The radially distributed array of electrospray nozzles 624 on amulti-system chip may be interfaced with a sampling orifice of a massspectrometer by positioning the nozzles near the sampling orifice. Thetight radial configuration of the electrospray nozzles 624 allows thepositioning thereof in close proximity to the sampling orifice of a massa spectrometer.

The multi-system chip 650 may be rotated relative to the samplingorifice to position one or more of the nozzles for electrospray near thesampling orifice. Appropriate voltage(s) may then be applied to the oneor more of the nozzles for electrospray. Alternatively, the multi-systemchip 650 may be fixed relative to the sampling orifice of a massspectrometer such that all nozzles, which converge in a relatively tightradius, are appropriately positioned for the electrospray process. As isevident, eliminating the need for nozzle repositioning allows for highlyreproducible and quick alignment of the single multi-system chip andincreases the speed of the analyses.

One, some or all of the radialily distributed nozzles 624 of theelectrospray devices 620 may generate electrosprays simultaneously,sequentially or randomly as controlled by the voltages applied to theappropriate electrodes of the electrospray device 620.

While specific and preferred embodiments of the invention have beendescribed and illustrated herein, it will be appreciated thatmodifications can be made without departing from the spirit of theinvention as found in the appended claims.

What is claimed and desired to be secured by United States LettersPatent is:
 1. An integrated chemical analysis system, comprising: afirst substrate having a major surface; a second substrate bonded to orotherwise attached to said first substrate; a liquid chromatographysystem integrated in said second substrate, and configured to receive afluid for analysis and to process and output the fluid; and a monolithicelectrospray device having an entrance orifice on an injection side anda nozzle on an ejection surface on an opposite planar side from theinjection side, a channel extending continuously through the monolithicdevice in communication with the entrance orifice and the nozzle, and aregion surrounding the nozzle recessed from the ejection surface,wherein said monolithic device is integrated on said first substrate,the injection side configured to receive the processed fluid from theliquid chromatography system, the major surface being configured todispense the fluid by electrospraying the fluid.
 2. The system of claim1, wherein the major surface of the monolithic electrospray device isconfigured to dispense the processed fluid by electrospraying the fluidin a direction generally perpendicular to the major surface.
 3. Thesystem of claim 1, wherein a plurality of said liquid chromatographysystems are each integrated in said second substrate, and eachconfigured to receive a fluid for analysis and to process and output thefluid; and a plurality of said monolithic electrospray devices, eachintegrated on said first substrate, the monolithic electrospray deviceseach having an injection side configured to receive the processed fluidfrom the liquid chromatography system, and each having an ejectionsurface configured to dispense the fluid by electrospraying the fluid.